Assays for detecting molecular modifications such as phosphate modifications and the presence and/or activity of enzymes and other agents involved in facilitating or otherwise regulating such modifications.
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1. A method of detecting the activity of an enzyme that performs a phosphate modification on a substrate to form a product in a sample, comprising:
contacting the substrate with the enzyme in the sample;
contacting the sample with a binding partner that specifically binds to the substrate or to the product, but not to both, wherein the binding partner includes Ga(III) ion that is required for binding between the binding partner and the substrate or the product;
detecting a response, based on luminescence polarization, indicative of the extent of binding between the substrate or the product and the binding partner without separating the bound substrate or product from the unbound substrate or product; and correlating the response with the activity of the enzyme.
2. The method of
exposing the sample to polarized light; and
measuring the degree of polarization of light emitted from the sample, in response to the step of exposing, wherein the degree of polarization is indicative of the extent of binding between the substrate or product and the binding partner.
3. The method of
4. The method of
6. The method of
7. The method of
8. The method of
10. The method of
11. The method of
12. The method of
13. The method of
14. The method of
15. The method of
16. The method of
contacting the substrate and enzyme with a candidate compound; and
determining the ability of the candidate compound to enhance or inhibit enzyme activity by its effects on the response.
17. The method of
18. The method of
providing a sample holder having a plurality of sample sites supporting a corresponding plurality of samples; and
repeating the steps of contacting, detecting, and correlating for each of the plurality of samples.
19. The method of
20. The method of
21. The method of
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This application is based upon and claims benefit under 35 U.S.C. §119(e) of the following U.S. provisional patent applications, which are incorporated herein by reference: Ser. Nos. 60/200,594, filed Apr. 28, 2000; Ser. No. 60/223,642, filed Aug. 8, 2000; and Ser. No. 60/241,032, filed Oct. 17, 2000.
This application is a continuation of and claims benefit under 35 U.S.C. §120 of the following patent applications, which are incorporated herein by reference: PCT Patent Application Ser. No. PCT/US00/16025, filed Jun. 9, 2000; and is a continuation of U.S. patent application Ser. No. 09/596,444, filed Jun. 19, 2000.
This application incorporates by reference the following U.S. patents: U.S. Pat. No. 5,843,378, issued Dec. 1, 1998; U.S. Pat. No. 6,965,381, issued Oct. 12, 1999; U.S. Pat. No. 6,071,748, issued Jun. 6, 2000; and U.S. Pat. No. 6,097,025, issued Aug. 1, 2000.
This application also incorporates by reference the following U.S. patent applications: Ser. No. 08/840,553, filed Apr. 14, 1997; Ser. No. 09/118,141, filed Jul. 16, 1998; Ser. No. 09/144,578, filed Aug. 31, 1998; Ser. No. 09/156,318, filed Sep. 18, 1998; Ser. No. 09/349,733, filed Jul. 8, 1999; Ser. No. 09/478,819, filed Jan. 5, 2000; Ser. No. 09/596,444, filed Jun. 19, 2000; Ser. No. 09/626,208, filed Jul. 26, 2000; Ser. No. 09/643,221, filed Aug. 18, 2000; Ser. No. 09/710,061, filed Nov. 10, 2000; Ser. No. 09/722,247, filed Nov. 24, 2000; Ser. No. 09/733,370, filed Dec. 8, 2000; Ser. No. 09/759,711, filed Jan. 12, 2001; Ser. No. 09/765,869, filed Jan. 19, 2001; Ser. No. 09/765,874, filed Jan. 19, 2001; Ser. No. 09/766,131, filed Jan. 19, 2001; Ser. No. 09/767,316, filed Jan. 22, 2001; Ser. No. 09/767,434, filed Jan. 22, 2001; Ser. No. 09/767,579, filed Jan. 22, 2001; Ser. No. 09/767,583, filed Jan. 22, 2001; Ser. No. 09/768,661, filed Jan. 23, 2001; Ser. No. 09/768,742, filed Jan. 23, 2001; Ser. No. 09/768,765, filed Jan. 23, 2001; Ser. No. 09/770,720, filed Jan. 25, 2001; Ser. No. 09/770,724, filed Jan. 25, 2001; Ser. No. 09/777,343, filed Feb. 5, 2001; Ser. No. 09/813,107, filed Mar. 19, 2001; Ser. No. 09/815,932, filed Mar. 23, 2001; and Ser. No. 09/836,575, filed Apr. 16, 2001 entitled Arc Lamp Power Supply, and naming David P. Stumbo as inventor.
This application also incorporates by reference the following U.S. provisional patent applications: Ser. No. 60/178,026, filed Jan. 26, 2000; Ser. Nos. 60/222,222, filed Aug. 1, 2000; Ser. No. 60/244,012, filed Oct. 27, 2000; Ser. No. 60/250,681, filed Nov. 30, 2000; Ser. No. 60/250,683, filed Nov. 30, 2000; and Ser. No. 60/267,639, filed Feb. 10, 2001.
This application also incorporates by reference the following publications: Richard P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (6th ed. 1996); Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy (2nd Edition 1999); and Bob Sinclair, Everything's Great When It Sits on a Chip: A Bright Future for DNA Arrays, 13 THE SCIENTIST, May 24, 1999, at 18.
The invention relates to assays for molecular modifications. More particularly, the invention relates to assays for detecting molecular modifications such as phosphate modifications and the presence and/or activity of enzymes and other agents involved in facilitating or otherwise regulating such modifications.
The physiological modification of molecules and supramolecular assemblies plays a major role in the structure and regulation of biological systems. These modifications may include phosphorylation, cyclization, glycosylation, acylation, and/or sulfation, among others, and the modified molecules may include polypeptides, nucleic acids, and/or lipids, among others. The importance of modifications is particularly evident in the cell-signaling pathway, in which extracellular and intracellular substances related by phosphate modifications such as phosphorylation and cyclization influence the position, nature, and activity of cells.
Signaling cells 102 are cells capable of producing a signal (substance) that can effect a specific response in another (target) cell. The signaling cells may be components of an endocrine, paracrine, or nervous system. The endocrine system is an organism-wide control system that regulates body function using hormones released by endocrine organs into the bloodstream. The endocrine organs include the pituitary gland, thyroid gland, parathyroid glands, adrenal glands, thymus gland, pineal body, pancreas, ovaries, testes, and kidneys. The paracrine system is a local control system that regulates nearby cells using local mediators released into the extracellular medium. The nervous system is a specialized control system that regulates specific cells using electrical impulses and neurotransmitters.
Signal substances 104a,b are substances through which a signaling cell may communicate with target cells, evoking a specific response. Signal substances may act as hormones, local mediators, and/or neurotransmitters, among others. Signal substances may take the form of proteins, small peptides, amino acids, nucleotides, steroids (e.g., cortisol, steroid sex hormones, vitamin D), retinoids, fatty acid derivatives, and dissolved gases (e.g., nitric oxide (NO) and carbon monoxide (CO)), among others.
Target cells 106 are cells capable of responding to a specific signal substance produced by a signaling cell. The ability to respond may depend on the cell and on the signal substance. For example, the signal substance thyroxine from the thyroid gland may evoke a response in nearly all cells, whereas the signal substance progesterone from the ovary may evoke a response only in specific cells in the lining of the uterus. The target response may include kinase activity, GTP binding, and/or cyclic nucleotide production.
The ability of a cell to respond to a given signal substance generally is determined by whether the cell includes a receptor for the signal substance. Here, a receptor is any molecule or supramolecular assembly capable of specifically binding a signal substance and initiating a response in a target cell. Representative receptors include cell-surface receptors 110 located on the surface of the target cell and intracellular receptors 112 located within the cytosol 114 or nucleus 116 of the target cell.
The nature of the response initiated by binding of a signal substance is determined by the intracellular machinery to which the receptor is operatively coupled. For example, binding of the neurotransmitter acetylcholine to identical receptors in heart muscle cells and secretory cells causes muscle relaxation in the heart muscle cells and secretion in the secretory cells, due to differences in the associated intracellular machinery.
The remainder of this section examines (1) the receptor mechanisms that cells use to bind signal substances and to communicate this binding to the cell interior, (2) the intracellular pathways that cells use for regulation, (3) the effects of errors in cell-signaling pathways, and (4) selected shortcomings of current cell-signaling assays.
1. Receptor Mechanisms
Target cells generally have receptors capable of specifically binding specific signal substances, including cell-surface receptors and/or intracellular receptors, as described above. Cell-surface receptors are more common and include (A) G-protein-linked receptors, (B) enzyme-linked receptors, and (C) ion-channel-linked receptors. These receptors typically bind large and/or water-soluble signal substances, such as many peptide hormones. Intracellular receptors are less common and include (A) guanylyl cyclase and (B) ligand-activated gene regulatory proteins. These receptors typically bind small and/or water-insoluble signal substances, such as steroid hormones, thyroid hormones, retinoids, vitamin D, and NO.
In the cAMP pathway, the target protein may be adenylyl cyclase (also known as adenylate cyclase), and the G-protein may be a stimulatory G-protein (Gs) that activates the adenylyl cyclase to make cAMP, or an inhibitory G protein (Gi) that inhibits the adenylyl cyclase to prevent it from making cAMP. The cAMP produced by the adenylyl cyclase activates cAMP-dependent protein kinase (A-kinase), which is a serine/threonine kinase that in turn activates or inhibits other enzymes to effect a physiological response. For example, in connection with glycogen metabolism, A-kinase may inhibit glycogen synthase to shut down glycogen synthesis, and simultaneously activate phosphorylase kinase that in turn activates glycogen phosphorylase to break down glycogen. A variety of signal substances use cAMP as a second messenger, including calcitonin, chorionic gonadotropin, corticotropin, epinephrine, follicle-stimulating hormone, glucagon, luteinizing hormone, lipotropin, melanocyte-stimulating hormone, norepinephrine, parathyroid hormone (PTH), thyroid-stimulating hormone, and vasopressin. The level of cAMP may be reduced by phosphodiesterases (PDEs), and the activity of kinases may be reversed by phosphatases, as described below.
In the Ca2+ pathway, the target protein may be a phospholipase with specificity for a phosphoinositide (i.e., inositol phospholipid), and the G-protein may be Gq, which activates the phospholipase to cleave the phosphoinositide to produce an intermediate that releases Ca2+ from the endoplasmic reticulum. For example, the phospholipase phosphoinositide-specific phospholipase C (phospholipase C-β) cleaves the phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the second messengers inositol triphosphate (IP3) and diacylglycerol. The inositol triphosphate is water soluble and diffuses to the endoplasmic reticulum (ER), where it releases Ca2+ from the ER by binding to IP3-gated Ca2+-release channels in the ER membrane. The diacylglycerol is membrane bound and may be cleaved to form the second messenger arachidonic acid or may activate the Ca2+-dependent serine/threonine kinase protein kinase C that in turn activates or inhibits other enzymes to effect a response. A variety of signal substances use Ca2+ as a second messenger, including acetylcholine, thrombin, and vasopressin.
The signal substance also may bind to intracellular receptors, such as guanylyl cyclase. This enzyme produces cGMP from GTP, which then acts as a second messenger much like cAMP. As described above, cGMP also may be produced by enzyme-linked cell-surface receptors. cGMP is present in most tissues at levels 1/10 to 1/100 those of cAMP. A variety of compounds increase cGMP levels in cells, including (1) the hormones acetylcholine, insulin, and oxytocin, (2) the guanylate cyclase stimulators (and vasodilators) nitroprusside, nitroglycerin, sodium nitrate, and nitric oxide, (3) chemicals such as serotonin and histamine, and (4) peptides such as atrial natriuretic peptide (ANP) that relax smooth muscle.
The level of cyclic nucleotides such as cAMP and cGMP may be controlled by specialized enzymes known as phosphodiesterases (PDEs). These enzymes catalyze the hydrolysis of the 3′-ester bond of cAMP and/or cGMP to form the corresponding uncyclized nucleotide monophosphates AMP and GMP, respectively. More than 30 human PDEs are known, and there is great interest in the pharmaceutical industry in finding inhibitors for PDEs for a broad range of potential therapeutic applications. A selective inhibitor of PDE-5 has been commercialized as the drug Viagra™ (i.e., Sildenafil) for the treatment of male erectile dysfunction. Moreover, several PDE-4 inhibitors are in clinical trials as anti-inflammatory drugs for the treatment of diseases such as asthma
2. Intracellular Signaling Pathways
Target cells may have intracellular signaling pathways capable of specifically binding signal substances, including cell-surface receptors and intracellular receptors, as described above. These pathways may include (1) a phosphorylation pathway involving ATP/ADP, and (2) a GTP-binding pathway involving GTP/GDP.
Specialized enzymes control phosphorylation in cells. In particular, protein kinase enzymes transfer phosphate groups to proteins, and protein phosphatase enzymes remove phosphate groups from proteins. Protein kinases and protein phosphatases are found in great variety in eucaryotic cells: a single cell may contain more than 100 different kinases, and one percent of genes may code for kinases.
There are two major categories of protein kinases: (1) serine/threonine (S/T) kinases, and (2) tyrosine kinases. The S/T kinases function by selectively phosphorylating serine and threonine side chains on substrate proteins or peptides. These kinases include cyclic AMP-dependent kinase (A-kinase), cyclic GMP-dependent kinase (G-kinase), protein kinase C (C-kinase), Ca2+-calmodulin-dependent kinase (CaM-kinase), phosphorylase kinase, MAP kinase, and TGF-β receptor, among others. The S/T kinases are predominantly cytosolic. The tyrosine kinases function by selectively phosphorylating tyrosine side chains on substrate proteins or peptides. These kinases include the receptor kinases for epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), insulin, insulinlike growth factor-1 (IGF-1), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and macrophage colony stimulating factor (M-CSF). These kinases also include the nonreceptor kinases associated with the tyrosine-kinase-associated receptors, such as the Src family (Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, and Blk) and Janus family (JAK1, JAK2, and Tyk2) kinases. The tyrosine kinases are predominantly membrane bound. A few kinases function by selectively phosphorylating threonine and tyrosine side chains on substrate proteins or peptides. These kinases include the mitogen-activated protein (MAP) kinase-kinase.
A physiological response may require stimulation by only a single type of signal substance, or may require stimulation by two or more types of signal substances. The latter mechanism permits finer tuning of the physiological response through signal integration. For example, a protein may be activated only by phosphorylation by two different kinases, themselves activated by binding of two different signal substances to two different receptors. Alternatively, a protein may be activated only by concurrent phosphorylation and GTP binding, or by binding of two subunits whose binding is contingent on phosphorylation by separately activated kinases.
3. Effects of Errors
Errors in the signal transduction and regulation pathways described above can cause cancer and other diseases. Indeed, a primary cause of cancer is a mutation that makes a stimulatory gene product hyperactive, converting a proto-oncogene into an oncogene. The primary classes of known proto-oncogenes include the following cell-signaling proteins: (1) growth-factor receptors acting via tyrosine kinases, (2) GTP binding proteins, (3) membrane/cytoskeleton-associated tyrosine kinases, (4) cytoplasmic tyrosine kinases, (5) steroid-type growth-factor receptors, and (6) S/T kinases. Consequently, cell-signaling proteins have become important subjects of research and drug development.
4. Selected Shortcomings of Current Assays
Assays that determine the presence and/or activity of cell-signaling components are important tools in life sciences research, including high-throughput screening. Unfortunately, current assays have a number of shortcomings.
The presence and activity of kinases, for example, can be determined using assays capable of detecting phosphorylated amino acids. In a standard kinase assay, radioactive ATP and an appropriate protein substrate are added to a sample. If the sample includes kinases, radioactive phosphate groups will be transferred from the radioactive ATP to the protein substrate. The presence and activity of kinases can be determined by assaying the amount of radioactive protein substrate, for example, using heterogeneous methods such as a filter plate that involve separating the protein substrate and radioactive ATP, or homogeneous methods such as a scintillation proximity assay for detecting radioactive decay. Unfortunately, both approaches involve radioactivity, presenting a short-term safety hazard for the assay operator and a long-term storage and disposal problem.
In an alternative kinase assay, ATP, a luminescent protein, and an antibody against a phosphorylated form of the luminescent protein are added to a sample. If the sample includes kinases, the kinases will transfer phosphate groups from the ATP to the protein, the antibody will bind to the phosphorylated protein, and the luminescence polarization of the protein will increase (because its rotational mobility will decrease). Unfortunately, the binding of antibodies is very target specific, so that in general a different antibody will be needed for each substrate (depending on the sequence of the substrate, including whether a tyrosine, serine, or threonine is to be phosphorylated). This shortcoming is especially significant for serines and threonines. Thus, a different antibody may be needed for each of the many kinases, depending on the polypeptide sequences of the corresponding substrates. Yet, suitable antibodies may be unavailable for many substrates and kinases, especially for poorly studied or previously unstudied kinases, or take several months or more to prepare.
Significantly, assays for other cell-signaling components (such as phosphatases, phosphodiesterases, and/or cyclases) may have similar shortcomings, such as the use of radioactive reagents, if the assays exist at all. Moreover, these assays may have slow time courses and unstable endpoints that require precise timing of assay readouts. Thus, there is a need for improved assays for detecting enzyme activity, and in particular the presence and activity of cell-signaling components.
The invention provides assays for detecting molecular modifications such as phosphate modifications and the presence and/or activity of enzymes and other agents involved in facilitating or otherwise regulating such modifications.
The various technical terms used herein generally have the meanings that are commonly recognized by those skilled in the art. However, the following terms may have additional and/or alternative meanings, as described below:
Cyclization/decyclization—the formation or degradation of a ring connecting a phosphate group and a nucleoside in a nucleotide. A common cyclization forms cAMP and cGMP from ATP and GTP, respectively, by removing two phosphate groups from the nucleotide triphosphates and joining the “free” end of the remaining phosphate group to the sugar in the remaining nucleotide monophosphate. A common decyclization reaction degrades the ring to form AMP and GMP from cAMP and cGMP, respectively.
Immunoglobulin—a group of typically large glycoproteins secreted by plasma cells in vertebrates that function as antibodies in the immune response by binding to specific antigens.
Luminescent—capable of, suitable for, or exhibiting luminescence, which is the emission of light by sources other than a hot, incandescent body. Luminescence is caused by electronic transitions within a luminescent substance (or luminophore) from more energetic to less energetic states. Among several types are chemiluminescence, electrochemiluminescence, electroluminescence, photoluminescence, and triboluminescence, which are produced by chemical reactions, electrochemical reactions, electric discharges, absorption of light, and the rubbing or crushing of crystals, respectively. Molecules may be intrinsically and/or extrinsically luminescent, meaning that they are luminescent on their own or luminescent due to covalent and/or noncovalent association with another molecule that is luminescent. Exemplary luminescent molecules and mechanisms for producing luminescent molecules are described in U.S. patent application Ser. No. 09/815,932, filed Mar. 23, 2001, which is incorporated herein by reference.
Nucleotide—a compound comprising a nucleoside and a phosphate group, some of which function as cell regulators and some of which function as the basic constituent of DNA and RNA. A nucleoside in turn is a compound comprising a sugar, such as ribose or deoxyribose, and a purine or pyrimidine base, such as adenine, cytosine, guanine, thymine, or uracil. Nucleotides are named according to the identities of their constituent bases and sugars, the number of their constituent phosphates, and the presence or absence of cyclization. Suitable nucleotides are listed in the following table:
Nucleotide
Abbreviation
Adenosine cyclic monophosphate
cAMP
Cytidine cyclic monophosphate
cCMP
Guanosine cyclic monophosphate
cGMP
Thymidine cyclic monophosphate
cTMP
Uridine cyclic monophosphate
cUMP
Adenosine monophosphate
AMP
Cytidine monophosphate
CMP
Guanosine monophosphate
GMP
Thymidine monophosphate
TMP
Uridine monophosphate
UMP
Phosphorylation/dephosphorylation—the introduction or removal of a phosphate group to or from an organic molecule such as a polypeptide. Phosphorylation is a versatile posttranslational modification that is a recurrent theme for regulation of enzyme activity and signal transduction pathways.
Polypeptide—a polymer comprising two or more amino acid residues linked together by covalent bonds, typically from amino end to carboxyl end by peptide bonds, and modifications and complexes thereof. Polypeptides generally include peptides and/or proteins, among others. Here, peptide generally refers to smaller polypeptides (e.g., less than about 100, 50, 20, or 10 amino acids, among others), and protein generally refers to larger polypeptides, and complexes thereof, possibly modified by other organic or inorganic conjugated chemical groups, such as phosphates, sugars, and so on. Polypeptides may include straight chains and/or branched chains, among others. Suitable amino acids are listed in the following table:
Three-letter
One-letter
Amino acid
abbreviation
abbreviation
Alanine
Ala
A
Arginine
Arg
R
Asparagine
Asn
N
Aspartic acid
Asp
D
Asparagine or aspartic acid
Asx
B
Cysteine
Cys
C
Glutamine
Gln
Q
Glutamic acid
Glu
B
Glutamine or glutamic acid
Glx
Z
Glycine
Gly
G
Histidine
His
H
Isoleucine
Ile
I
Leucine
Leu
L
Lysine
Lys
K
Methionine
Met
M
Phenylalanine
Phe
F
Proline
Pro
P
Serine
Ser
S
Threonine
Thr
T
Tryptophan
Trp
W
Tyrosine
Tyr
Y
Valine
Val
V
Specific binding—binding to a specific binding partner to the exclusion of binding to most other moieties. Specific binding can be characterized by a binding coefficient. Generally, specific binding coefficients range from 10−4 M to 10−12 M and lower, and preferred specific binding coefficients range from 10−8 or 10−9 M to 10−12 M and lower.
The invention provides assays for detecting molecular modifications and the presence and/or activity of enzymes and other agents involved in facilitating or otherwise regulating such modifications. The modifications may include among others phosphate modifications, such as the phosphorylation and dephosphorylation of molecules such as polypeptides and the cyclization and decyclization of molecules such as nucleotides. The enzymes may include among others enzymes involved in performing and/or regulating phosphorylation, dephosphorylation, cyclization, and decyclization modifications, such as kinases, phosphatases, cyclases, and phosphodiesterases (PDEs), respectively. The assays may include among others luminescence assays, such as luminescence polarization, luminescence resonance energy transfer, and/or luminescence intensity. The assays provided by the invention may be useful in a variety of applications, including without limitation life science research, drug research, accelerated drug discovery, assay development, and high-throughput screening, among others.
A and A* generally comprise any two species related by a modification (denoted by the presence or absence of *). A and A* may include molecules and assemblies of molecules such as polypeptides and/or nucleotides, among others. The modification may include phosphate modifications such as phosphorylation, dephosphorylation, cyclization, and/or decyclization, among others, and nonphosphate modifications such as nonphosphate posttranslational modifications of polypeptides, among others. A and A* may be related as substrate and product in a reaction, such as an enzyme-catalyzed reaction. Thus, depending on the direction of the reaction, A and A* in a phosphate modification may be a phosphorylated polypeptide, a nonphosphorylated polypeptide, a cyclized nucleotide, or a noncyclized nucleotide, among others. In some embodiments, A and/or A* may include components intended to facilitate detection of binding between A or A* and BP, such as a luminophore, a quencher, an energy transfer partner, and the like.
BP generally comprises any binding partner capable of binding specifically to A or A* (i.e., the modified species or the unmodified species) but not to both. BP may include any binding partner having the specified binding properties that does not include a polypeptide and/or an immunoglobulin, and/or a functional portion or fragment thereof. Alternatively, or in addition, BP may include one or more metal ions, including dicationic, tricationic, and polycationic metal ions, among others. Suitable dicationic metal ions include iridium (Ir2+), osmium (OS2+), platinum (Pt2+), rhenium (Re2+), and ruthenium (Ru2+), among others. Suitable tricationic metal ions include including aluminum (Al3+), chromium (Cr3+), iron (Fe3+), gallium (Ga3+), manganese (Mn3+), scandium (Sc3+), titanium (Ti3+), vanadium (V3+), and/or yttrium (Y3+), among others. Preferred metal ions include aluminum, iron, and gallium. The metal ions may interact with or otherwise be involved in or required for binding with the modification on A or A*, such as the phosphate group on a phosphorylated protein or a noncyclized nucleotide. Alternatively, or in addition, BP may include one or more charged portions to facilitate or otherwise participate in the binding reaction with A or A*, particularly charged portions that are immobilized relative to BP. Alternatively, or in addition, BP may bind to a substrate such as A or A* only if it is phosphorylated, where the binding between the substrate and the binding partner is substantially nonspecific with respect to the structure of the substrate aside from any phosphate groups. Thus, the binding may occur substantially without regard to the target amino acid or surrounding amino acid sequence in a phosphorylation/dephosphorylation assay, or the base or nucleoside in a cyclization/decyclization assay. Alternatively, or in addition, BP may include a macromolecule and/or a particle. Here, particles include nanoparticles and microparticles, among others, where nanoparticles are particles with at least one dimension less than about 100 nm, and microparticles are particles with dimensions between about 100 nm and about 10 μm. Alternatively, or in addition, BP may be linked to an associated solid phase, such as a bead, membrane, or sample holder, among others. The link may be formed using any suitable mechanism, including hydrogen bonding, ionic bonding, electrostatic binding, hydrophobic interactions, Van der Waals interactions, and/or covalent attachment, among others. In some embodiments, BP may include components intended to facilitate detection of binding between BP and A or A*, such as a luminophore, a quencher, an energy transfer partner, and the like.
EAA* and EA*A generally comprise any enzymes or other catalysts capable of facilitating reactions converting A to A* and A* to A, respectively. EAA* and EA*A may include among others enzymes such as kinases and phosphatases, which catalyze the addition and removal of phosphate groups to and from polypeptides, respectively. EAA* and EA*A also may include enzymes such as cyclases and phosphodiesterases, which catalyze the cyclization and decyclization of nucleotides, respectively.
MEAA* and MEA*A generally comprise any modulators or other agents capable of modulating or otherwise affecting the activity of EAA* and EA*A, respectively. The modulator may be a change in environmental condition, such as a change in sample temperature, but more typically is an enzyme or other reagent added to the sample. The modulator may be a chemical reagent, such as an acid, base, metal ion, organic solvent, and/or other substance intended to effect a chemical change in the sample. Alternatively, or in addition, the modulator may have or be suspected to have a biological activity or type of interaction with a given biomolecule, such as an enzyme, drug, oligonucleotide, nucleic acid polymer, peptide, protein, and/or other biologically active molecule. The modulator may include an agonist or inhibitor capable of promoting or inhibiting, respectively, the activity of the modulated enzyme. For example, in a cyclic nucleotide assay, preferred agonists include forskolin and isoproterenol, and preferred inhibitors include propranolol and Zaprinast.
1. Contacting
The step of contacting assay components such as enzymes, enzyme modulators, substrates, products, and/or binding partners with one another and/or with other species generally comprises any method for bringing any specified combination of these components into functional and/or reactive contact. A preferred method is by mixing and/or forming the materials in solution, although other methods, such as attaching one or more components such as the binding partner to a bead or surface, also may be used, as long as the components retain at least some function, specificity, and/or binding affinity following such attachment. Exemplary apparatus having fluidics capability suitable for contacting or otherwise preparing assay components are described in the following patent applications, which are incorporated herein by reference: U.S. patent application Ser. No. 09/777,343, filed Feb. 5, 2001; and U.S. Provisional Patent Application Ser. No. 60/267,639, filed Feb. 10, 2001.
One of more of the assay components may comprise a sample, which typically takes the form of a solution containing one or more biomolecules that are biological and/or synthetic in origin. The sample may be a biological sample that is prepared from a blood sample, a urine sample, a swipe, or a smear, among others. Alternatively, the sample may be an environmental sample that is prepared from an air sample, a water sample, or a soil sample, among others. The sample typically is aqueous but may contain biologically compatible organic solvents, buffering agents, inorganic salts, and/or other components known in the art for assay solutions.
The assay components and/or sample may be supported for contact and/or analysis by any substrate or material capable of providing such support. Suitable substrates may include microplates, PCR plates, biochips, and hybridization chambers, among others, where features such as microplate wells and microarray (i.e., biochip) sites may comprise assay sites. Suitable microplates are described in the following U.S. patent applications, which are incorporated herein by reference: Ser. No. 08/840,553, filed Apr. 14, 1997; and Ser. No. 09/478,819, filed Jan. 5, 2000. These microplates may include 96, 384, 1536, or other numbers of wells. These microplates also may include wells having small (≦50 μL) volumes, elevated bottoms, and/or frusto-conical shapes capable of matching a sensed volume. Suitable PCR plates may include the same (or a similar) footprint, well spacing, and well shape as the preferred microplates, while possessing stiffness adequate for automated handling and thermal stability adequate for PCR. Suitable microarrays include nucleic acid and polypeptide microarrays, which are described in Bob Sinclair, Everything's Great When It Sits on a Chip: A Bright Future for DNA Arrays, 13 THE SCIENTIST, May 24, 1999, at 18, which is incorporated herein by reference: Suitable hybridization chambers are described in PCT Patent Application Ser. No. PCT/US99/03678, filed Feb. 19, 1999, which is incorporated herein by reference.
2. Detecting
The step of detecting a response indicative of the extent of binding generally comprises any method for effectuating such detection, including detecting and/or quantifying a change in, or an occurrence of, a suitable parameter and/or signal. The method may include luminescence and/or nonluminescence methods, and heterogeneous and/or homogeneous methods, among others.
Luminescence and nonluminescence methods may be distinguished by whether they involve detection of light emitted by a component of the sample. Luminescence assays involve detecting light emitted by a luminescent compound (or luminophore) and using properties of that light to understand properties of the compound and its environment. A typical luminescence assay may involve (1) exposing a sample to a condition capable of inducing luminescence from the sample, and (2) measuring a detectable luminescence response indicative of the extent of binding between the member of interest and a corresponding binding partner. Most luminescence assays are based on photoluminescence, which is luminescence emitted in response to absorption of suitable excitation light. However, luminescence assays also may be based on chemiluminescence, which is luminescence emitted in response to chemical excitation, and electrochemiluminescence, which is luminescence emitted in response to electrochemical energy. Suitable luminescence assays include, among others, (1) luminescence intensity, which involves detection of the intensity of luminescence, (2) luminescence polarization, which involves detection of the polarization of light emitted in response to excitation by polarized light, and (3) luminescence energy transfer, which involves detection of energy transfer between a luminescent donor and a suitable acceptor. These and other luminescence assays are described below in Example 14 and materials cited therein. Nonluminescence assays involve using a detectable response other than light emitted by the sample, such as absorption, scattering, and/or radioactivity, among others. These and other nonluminescence assays are described in the following materials, which are incorporated herein by reference: U.S. patent application Ser. No. 09/765,869, filed Jan. 19, 2001; and Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy (2nd ed. 1999).
The detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal that is detectable by direct visual observation and/or by suitable instrumentation. Typically, the detectable response is a change in a property of the luminescence, such as a change in the intensity, polarization, energy transfer, lifetime, and/or excitation or emission wavelength distribution of the luminescence. The detectable response may be simply detected, or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property. In luminescence assays, the detectable response may be generated directly using a luminophore associated with an assay component actually involved in binding such as A* or BP, or indirectly using a luminophore associated with another (e.g., reporter or indicator) component. Suitable methods and luminophores for luminescently labeling assay components are described in the following materials, which are incorporated herein by reference: Richard P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (6th ed. 1996); U.S. patent application Ser. No. 09/813,107, filed Mar. 19, 2001; and U.S. patent application Ser. No. 09/815,932, filed Mar. 23, 2001.
Heterogeneous and homogeneous methods may be distinguished by whether they involve sample separation before detection. Heterogeneous methods generally require bulk separation of bound and unbound species. This separation may be accomplished, for example, by washing away any unbound species following capture of the bound species on a solid phase, such as a bead or microplate surface labeled with a tricationic metal ion or other suitable binding partner. The extent of binding then can be determined directly by measuring the amount of captured bound species and/or indirectly by measuring the amount of uncaptured unbound species (if the total amount is known). Homogeneous methods, in contrast, generally do not require bulk separation but instead require a detectable response such as a luminescence response that is affected in some way by binding or unbinding of bound and unbound species without separating the bound and unbound species. Homogeneous assays typically are simpler to perform but more complicated to develop than heterogeneous assays.
3. Correlating
The step of correlating generally comprises any method for correlating the extent of binding with the extent of modification of the assay component being analyzed, and/or with the presence and/or activity of an enzyme that affects the modification. The nature of this step depends in part on whether the detectable response is simply detected or whether it is quantified. If the response is simply detected, it typically will be used to evaluate the presence of a component such as a substrate, product, and/or enzyme, or the presence of an activity such as an enzyme or modulator activity. In contrast, if the response is quantified, it typically will be used to evaluate the presence and/or quantity of a component such as a substrate, product, and/or enzyme, or the presence and/or activity of a component such as an enzyme or modulator.
The correlation generally may be performed by comparing the presence and/or magnitude of the response to another response (e.g., derived from a similar measurement of the same sample at a different time and/or another sample at any time) and/or a calibration standard (e.g., derived from a calibration curve, a calculation of an expected response, and/or a luminescent reference material). Thus, for example, in a polarization assay for cyclic nucleotide concentration, the cyclic nucleotide concentration in an unknown sample may be determined by matching the polarization measured for the unknown with the cyclic nucleotide concentration corresponding to that polarization in a calibration curve generated under similar conditions by measuring polarization as a function of cyclic nucleotide concentration. More generally, the following table shows representative qualitative changes in the indicated detectable luminescence response upon binding between A* and BP following a forward reaction A→A*.
Intensity
Intensity
FP
ET
Label on A*
Label on BP
(Luminophore)
(Acceptor)
(Luminophore)
(Lum. → Acc.)
Luminophore
—
Increases
—
Luminophore
Increases
Luminophore
Quencher
Decreases
Quencher
Luminophore
Decreases
Luminophore
Acceptor
Decreases
Increases
Decreases
Increases
Acceptor
Luminophore
Decreases
Increases
Increases
This reaction is representative of a phosphorylation reaction performed by a kinase or a decyclization reaction performed by a PDE, assuming that the binding partner binds to the (noncyclized) phosphorylated species. Similarly, the following table shows representative qualitative changes in the indicated detectable luminescence response upon binding of A* and BP following the reverse reaction A*→A.
Intensity
Intensity
FP
ET
Label on A*
Label on BP
(Luminophore)
(Acceptor)
(Luminophore)
(Lum. → Acc.)
Luminophore
—
Decreases
—
Luminophore
Decreases
Luminophore
Quencher
Increases
Quencher
Luminophore
Increases
Luminophore
Acceptor
Increases
Decreases
Increases
Decreases
Acceptor
Luminophore
Increases
Decreases
Decreases
This reaction is representative of a dephosphorylation reaction performed by a phosphatase or a cyclization reaction performed by a cyclase, assuming again that the binding partner binds to the (noncyclized) phosphorylated species.
The following examples describe without limitation further aspects of the invention. These aspects include (1) the ability of binding partners such as tricationic metal ions to bind specifically to phosphorylated species such as phosphopeptides and nucleotides, and (2) the use of such binding in assays for enzymes and other agents involved in phosphorylation and dephosphorylation (e.g., kinases and phosphatases, respectively) and cyclization and decyclization (e.g., cyclases and PDEs, respectively), among others. These aspects are applicable to a wide variety of enzymes and enzyme substrates and products.
This example describes a macromolecular trapping system for use in luminescence polarization and/or energy transfer assays, among others, in accordance with aspects of the invention. In this system, Ru2+ is entrapped in small (˜20-30 kDa) synthetic polymer macromolecules (MM), which are obtained from PreSens Precision Sensing (Neuburg/Donau, Germany). These macromolecules are relatively hydrophilic, with carboxyl groups on their surfaces for activation. The MM with the entrapped Ru2+ is used as a support to immobilize tricationic metal cations, including Fe3+ and Ga3+. Specifically, the chelator imidodiacetic (IDA) acid is linked to the MM using the secondary amine group of IDA and a carboxyl group on the MM. Afterwards, the MM-IDA is incubated with either FeCl3 or GaCl3. The FeCl3 quenches the luminescence of Ru2+, whereas the GaCl3 does not. The macromolecule loaded with Fe3+ or Ga3+ is denoted MM-Fe or MM-Ga, respectively.
The macromolecular trapping system may be used in a variety of kinase, phosphatase, phosphodiesterase, and/or cyclase assays, as described below in Examples 3-5 and 7-9. In an exemplary assay, a kinase enzyme phosphorylates a luminescently labeled kinase substrate, which binds to the metal cations immobilized on the MM. Binding is detected using polarization and/or energy transfer methods, among others, for example, using apparatus and methods as described herein. Binding is detectable using polarization because binding leads to a decrease in substrate mobility and a concomitant increase in the polarization of light emitted by luminophores bound to the substrate. Similarly, binding is detectable using energy transfer because binding leads to a decrease in separation between the luminophores bound to the substrate and the Ru2+ immobilized in the MM, and a concomitant increase in energy transfer from the Ru2+ (donor) to the luminophore (acceptor).
This approach may be extended by various modifications and/or substitutions. For example, in polarization assays, the Ru2+ may be omitted, if desired. In energy transfer assays, the Ru2+ may be replaced by any energy transfer partner, as long as the energy transfer partner supported by the MM is capable of energy transfer to or from a complementary energy transfer partner supported by the species binding to the MM. Exemplary energy transfer partners are described in U.S. patent application Ser. No. 09/815,932, filed Mar. 23, 2001, which is incorporated herein by reference. Also, the Ru2+ or its analog does not need to be encapsulated in the MM. A luminescent species may be attached directly to a suitable Fe3+ or Ga3+ chelate. In a heterogeneous assay, phosphorylated proteins bound via Ga3+ or Fe3+ to microplates, particles, or inner surfaces of microfluidic devices may be detected after a wash by measuring luminescence intensity. Such detection can take place either directly on the surfaces or in the solution phase by adding an elution solution such as a phosphate buffer. With other detection methods, such as the laser-scanning method used in fluorometric microvolume assay technology (FMAT™) technology (PE Biosystems, Foster City, Calif.), the bound phosphoproteins can be detected directly on the beads, without the need for washing or separation. Other labels such as enzymes also may be used in the heterogeneous format.
Potential difficulties with this system include (1) interference from compounds (e.g., ATP, free phosphate, EDTA, and possibly primary/secondary amines) that may compete with or otherwise affect the interaction between the metal and the phosphorylated protein, and (2) difficulty in maintaining a pH that preserves the affinity and selectivity of the binding between the metal and phosphorylated protein.
This example describes assays for the presence, activity, substrates, and/or products of kinases in accordance with aspects of the invention. Similar assays may be used to analyze phosphatases in which the substrates and products of the kinase reaction become the products and substrates of the phosphatase reaction, respectively.
Kinases catalyze the addition of phosphate groups to appropriate substrates, as shown below:
##STR00001##
Thus, the presence and/or activity of a kinase may be detected by a decrease in the concentration of a nonphosphorylated (e.g., polypeptide) substrate and/or by an increase in the concentration of a corresponding phosphorylated product, among others. (The presence and/or activity of a phosphatase may be detected similarly by a decrease in the concentration of a phosphorylated substrate and/or an increase in the concentration of a nonphosphorylated product.) The invention provides among others kinase assays that involve contacting a sample containing a candidate kinase (and optionally a modulator thereof) with a luminescently labeled nonphosphorylated polypeptide having at least one amino acid subject to phosphorylation (such as a tyrosine, serine, and/or threonine) and a binding partner that binds specifically to the phosphorylated polypeptide but not to the nonphosphorylated polypeptide. These assays further involve detecting a response indicative of the extent of binding between the polypeptide and the binding partner such as luminescence intensity, polarization, and/or energy transfer, and correlating the response with the extent of phosphorylation or nonphosphorylation of the polypeptide, and thus with the activity of the candidate kinase. The binding partner may include a metal ion such as a tricationic metal ion that interacts with the phosphate group on the phosphorylated polypeptide to facilitate the binding reaction. The binding partner also may include a macromolecule, a nanoparticle, a solid phase portion, a quencher, and/or an energy transfer partner complementary to the luminophore on the polypeptide, depending in part on the detection scheme.
This example describes experiments to characterize binding between MM-Ga and a fluorescein-labeled di-phosphotyrosine 15-amino-acid peptide tracer denoted tyrosine kinase 1 (TK-1) tracer. These experiments show the utility of the MM-Ga system for detection of phosphorylated tyrosine and the presence and/or activity of tyrosine kinases and phosphatases.
This example describes experiments to characterize binding between MM-Ga and the following mono-seine fluorescein-labeled peptide tracer:
fluorescein-Leu-Arg-Arg-Ala-Ser-Leu-Gly (SEQ ID NO: 1)
This peptide is termed a “Kemptide,” and the fluorescein-labeled peptide is termed a “fluo-Kemptide.” These experiments use cAMP-dependent protein kinase A (PKA, Promega) as the enzyme and fluo-Kemptide as the substrate. These experiments show the utility of the MM-Ga system for detection of phosphorylated serine and the presence and/or activity of serine/threonine kinases and phosphatases.
Examples 3 and 4 describe homogeneous assays in which metal ions (e.g., Ga3+) immobilized on macromolecules system bind selectively to phosphorylated peptides generated in a kinase reaction. These assays may be used to monitor the time course and/or end point of a kinase (and/or phosphatase) reaction using various luminescence methods, including luminescence polarization.
This example describes a heterogeneous kinase assay in accordance with aspects of the invention. Here, the feasibility of using metal-coated plates in the development of generic kinase assays is demonstrated with a commercial Ni2+-coated plate (Pierce, Rockford, Ill.), in which the Ni2+ is replaced with Ga3+. Specifically, 200 μL of a 0.5 M EDTA-containing solution is added to each well of a 96-well Ni2+-coated plate, and the plate is incubated at room temperature for 1 hour. The process is repeated two more times to remove at least substantially all of the Ni2+ from the plate. The plate then is washed 3 times with 10 mM Tris buffer (pH 7.4). Next, 200 μL of a 0.1 M GaCl3 solution is added to each well of the plate, and the plate is incubated overnight at room temperature. The plate is washed three times before being used in a kinase assay. This procedure effectively converts the walls of the plate into assay surfaces capable of binding a phosphorylated substrate but not a nonphosphorylated substrate.
These experiments show the viability of a heterogeneous assay format and the specificity of binding to the tricationic versus dicationic metal ion. The heterogeneous assay format offers many of the advantages of the homogenous assays, including its applicability in principle to any kinase regardless of its substrate specificity. This may save assay developers 3 to 6 months of time and effort in making antibodies that recognize specifically a phosphorylated version of an amino acid sequence. The lack of availability of such special antibodies often is the major obstacle in the development of nonradioactive kinase assays. The heterogeneous assay format also allows for simple detection using luminescence intensity, without requiring polarizers or selection of complementary energy transfer pairs.
This example describes assays for the presence, activity, substrates, and/or products of phosphodiesterases in accordance with aspects of the invention. Similar assays may be used to analyze cyclases in which the substrate of the phosphodiesterase reaction becomes the product of the cyclase reaction.
Phosphodiesterases (PDEs) catalyze the decyclization of cyclic nucleotides to the corresponding noncyclized nucleotide monophosphate, as shown below:
##STR00002##
Thus, the presence and/or activity of a PDE may be detected by a decrease in the concentration of a cyclic nucleotide (cNMP) substrate and/or by an increase in the concentration of a corresponding uncyclized nucleotide monophosphate (NMP) product. (The presence and/or activity of a cyclase may be detected similarly by a decrease in the concentration of a nucleotide triphosphate substrate and/or by an increase in the concentration of a corresponding cyclic nucleotide.) The invention provides among others PDE assays that involve contacting a sample containing a candidate PDE (and optionally a modulator thereof) with a luminescently labeled cyclic nucleotide and a binding partner that binds specifically to the corresponding uncyclized nucleotide monophosphate but not to the cyclic nucleotide. The binding partner may include one or more of the attributes described above, such as a tricationic metal M3+ (e.g., Al3+, Ga3+, and/or Fe3+) capable of binding an uncyclized phosphate group but not a cyclized phosphate group, and optionally an energy transfer partner and/or quencher. PDE activity may be detected by an increase in NMP binding using any technique capable of measuring such an increase, including luminescence polarization, luminescence resonance energy transfer, luminescence intensity, and/or nonluminescence and/or heterogeneous techniques, among others. For example, PDE activity may be detected following NMP binding by (1) an increase in luminescence polarization (assuming that the lifetime and rotational correlation time of the binding partner are selected so that binding of the NMP to the binding partner measurably decreases the rotational correlation time of the NMP), (2) an increase in luminescence resonance energy transfer (assuming that the binding partner is associated with a suitable energy transfer partner), and/or (3) a decrease in luminescence intensity (assuming that the binding partner is associated with a suitable luminescence quencher).
The assays may include (1) contacting a sample containing a candidate PDE (and/or other cell-signaling component) with a luminescently labeled cyclic nucleotide and a binding partner capable of distinguishing between the cyclic nucleotide and the corresponding nucleotide monophosphate, (2) illuminating the sample with light capable of inducing luminescence in the sample, (3) measuring a property of the luminescence transmitted from the sample, and (4) correlating the property with the presence and/or activity of the cyclic nucleotide and/or the corresponding nucleotide monophosphate and hence the presence and/or activity of an associated enzyme.
The invention also provides methods for identifying modulators such as agonists and inhibitors of receptors and/or enzymes involved in the production and/or regulation of cell-signaling molecules, such as the hydrolysis of cyclic nucleotides. The methods may include looking for the effects of a modulator by conducting a method for determining the concentration of a cyclic nucleotide and/or the corresponding nucleotide monophosphate in both the presence and absence of the putative modulator. For example, in a polarization assay in which PDE activity leads to an increase in polarization, a decrease in the measured extent of polarization of the emitted light in the presence of the putative modulator identifies the putative modulator as an inhibitor of the receptor or enzyme, and an increase in the measured extent of polarization in the presence of the putative modulator identifies the putative modulator as an agonist of the receptor or enzyme.
This example describes end-point and time-course assays for PDE 5 in accordance with aspects of the invention, showing in part the utility of the MM-Ga system in PDE assays. These assays use the following components, among others: (1) cGMP-specific PDE (type V, Calbiochem, La Jolla, Calif.), (2) N-methylanthraniloyl (MANT) cGMP substrate (Molecular Probes, Eugene, Oreg.), and (3) MM-Ga, as described in Example 1. MANT is a compact blue-fluorescing luminophore that attaches to the cGMP via the ribose ring of the cGMP. These assays show the utility of the MM-Ga system for detection of noncyclized GMP and the presence and/or activity of cGMP-specific PDEs.
This example describes alternative PDE assays in accordance with aspects of the invention. These assays are presented in a homogenous, nonradioactive format using a carboxyfluorescein labeled cGMP substrate. The assay also may be used in a heterogeneous format and/or with an alternative luminescent cGMP and/or cAMP. These assays further show the utility of the MM-Ga system for detection of noncyclized GMP and the presence and/or activity of cGMP-specific PDEs, including the use of a different luminophore than the MANT of Example 7.
This example describes several aspects of the invention, including (1) use of Ga3+-coated nanoparticles as the binding component in the assay, (2) applications to the detection of PDE 4 enzyme, with fluorescein-labeled cAMP as substrate, and (3) applications to the detection of PDE 1 enzyme, with both fluorescein-labeled cAMP and fluorescein-labeled cGMP as substrates.
As discussed in Example 1, synthetic polymer macromolecules (MM) can be substituted with other materials that have a high molecular weight and that tricationic cations (i.e., Fe3+, Ga3+) can be immobilized on. Here, we use selected nanoparticles, including polystyrene nanoparticles having an average diameter of about 40 nm. The nanoparticles are from Bangs Laboratory (Fisher, Ind.), and are modified after acquisition from the vendor to attach Ga3+ on the surfaces of the particles.
This example shows representative tracers for use in cyclic nucleotide assays, particularly luminescence-polarization-based cyclic nucleotide assays. General structures for such tracers are shown below for (A) cAMP and (B) cGMP:
##STR00003##
Here, X and R1 represent linkers, which optionally and independently may be present or absent, and F1 represents a reporter species. X may include among others any alkyl, allyl, or aryl linker with ester or ether bonds to the cyclic nucleotide, including —OC(═O)—CH2CH2C(═O)—. R1 may be any linker joining FL to the nucleotide, directly, or indirectly through X, including a rigid linker having (two) reactive groups for coupling, one to FL and one to the nucleotide. For example, R1 may be a diamino-alkyl, -cycloalkyl, -aryl, or -allyl group, or a dihydroxy group that forms an amide or ester, respectively, with the groups X and F1. F1 may include any suitable reporter species, such as a luminophore for luminescence assays or an isotope for radioassays. For example, F1 may include a fluorescein or rhodamine that forms a thiourea, ester, or amide bond with the group X. Preferred structures include 1,2 and 1,4-diaminocyclohexyl-linked tracers, as described in U.S. patent application Ser. No. 09/768,661, filed Jan. 23, 2001, which is incorporated herein by reference.
This example describes methods and kits for detecting phosphate modifications and/or associated enzymes and modulators in whole cells. The methods generally comprise growing cells under desired conditions, lysing the cells, incubating the cells before and/or after lysis with one or more reagents, and detecting the presence, quantity, and/or activity of species and/or reactions of interest. The kits generally comprise collections of reagents and/or other materials of interest, including substrates, binding partners, and/or lysis buffers, among others. The methods and kits are described greater detail in the context of cyclic nucleotide assays for adherent and suspended cells in Examples 8 and 9 of U.S. patent application Ser. No. 09/768,661, filed Jan. 23, 2001, which is incorporated herein by reference.
This example describes miscellaneous applications and other uses for the various assays described herein.
The applications include detecting any of the modifications, enzymes, and/or modulators identified herein and/or in the following U.S. patent applications, which are incorporated herein by reference: Ser. No. 09/768,661, filed Jan. 23, 2001; and Ser. No. 09/596,444, filed Jun. 19, 2000. The modifications include phosphorylation, dephosphorylation, cyclization, and/or decyclization, among others, as described above. The enzymes include kinases, phosphatases, cyclases, and/or phosphodiesterases, among others, including variants such as isoenzymes thereof. For example, the cyclases include adenylyl cyclase and guanylyl cyclase, among others, and the phosphodiesterases include PDE 1 through PDE 10, among others. The modulators include modulators of these enzymes, among others. For example, the cyclase modulators include forskolin and ODQ, among others, and the phosphodiesterase modulators include cilostamide, dipyridamole, EHNA hydrochloride, etazolate hydrochloride, MBCQ, MMPX, MY-5445, Ro 20-1724, rolipram, siguazodan, vinpocetine, and Zaprinast, among others.
The applications also include combining assays for different modifications, enzymes, and/or modulators to form integrated assays, for example, by combining a phosphorylation assay and a cyclization assay to study signaling mechanisms involving multiple cell-signaling pathways.
This example describes kits for use in performing assays in accordance with aspects of the invention. The kits may include substrates and/or binding partners for performing the assays described herein. These substrates and/or binding partners may include luminophores, quenchers, and/or energy transfer partners, among others. The kits also may include sample holders such as microplates or biochips that have been treated to act as binding partners. The kits optionally may include additional reagents, including but not limited to buffering agents, luminescence calibration standards, enzymes, enzyme substrates, nucleic acid stains, labeled antibodies, or other additional luminescence detection reagents. The substrates, binding partners, and/or additional reagents optionally are present in pure form, as concentrated stock solutions, or in prediluted solutions ready for use in the appropriate energy transfer assay. Typically, the kit is designed for use in an automated and/or high-throughput assay, and so is designed to be fully compatible with microplate readers, microfluidic methods, and/or other automated high-throughput methods.
This example describes exemplary luminescence assays. Further aspects of these assays as well as additional luminescence assays and apparatus for performing luminescence assays are described in the following materials, which are incorporated herein by reference: U.S. Pat. No. 6,097,025, issued Sep. 24, 1998; U.S. patent application Ser. No. 09/349,733, filed Jul. 8, 1999; U.S. Provisional Patent Application Ser. No. 60/267,639, filed Feb. 10, 2001; and Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy (2nd ed. 1999).
Luminescence, as defined above, is the emission of light from excited electronic states of atoms or molecules, including photoluminescence, chemiluminescence, and electrochemiluminescence, among others. Luminescence may be used in a variety of assays, including (A) intensity assays, (B) polarization assays, and (C) energy transfer assays, among others.
A. Intensity Assays
Luminescence intensity assays involve monitoring the intensity (or amount) of light emitted from a composition. The intensity of emitted light will depend on the extinction coefficient, quantum yield, and number of luminescent analytes in the composition, among others. These quantities, in turn, will depend on the environment of the analyte, among others, including the proximity and efficacy of quenchers and energy transfer partners. Thus, luminescence intensity assays may be used to study binding reactions, among other applications.
B. Polarization Assays
Luminescence polarization assays involve the absorption and emission of polarized light. Here, polarization refers to the direction of the light's electric field, which generally is perpendicular to the direction of the light's propagation. In a luminescence polarization assay, specific molecules within a composition are labeled with one or more luminophores. The composition then is illuminated with polarized excitation light, which preferentially excites luminophores having absorption dipoles aligned parallel to the polarization of the excitation light. These molecules subsequently decay by preferentially emitting light polarized parallel to their emission dipoles. The extent of polarization of the total emitted light depends on the extent of molecular reorientation during the time interval between luminescence excitation and emission, which is termed the luminescence lifetime, τ. In turn, the extent of molecular reorientation depends on the luminescence lifetime and the size, shape, and environment of the reorienting molecule. Thus, luminescence polarization assays may be used to quantify binding reactions and enzymatic activity, among other applications. In particular, molecules commonly rotate (or “tumble”) via diffusion, with a rotational correlation time τrot that is proportional to their volume, or the cube of their radius of gyration. (This cubic dependence on radius makes polarization assays very sensitive to binding.) Thus, during their luminescence lifetime, relatively large molecules will not reorient significantly, so that their total luminescence will be relatively polarized. In contrast, during the same time interval, relatively small molecules will reorient significantly, so that their total luminescence will be relatively unpolarized.
The relationship between polarization and intensity is expressed by the following equation:
Here, P is the polarization, I∥ is the intensity of luminescence polarized parallel to the polarization of the excitation light, and I195 is the intensity of luminescence polarized perpendicular to the polarization of the excitation light. P generally varies from zero to one-half for randomly oriented molecules (and zero and one for aligned molecules). If there is little rotation between excitation and emission, I81 will be relatively large, I⊥ will be relatively small, and P will be close to one-half. (P may be less than one-half even if there is no rotation; for example, P will be less than one-half if the absorption and emission dipoles are not parallel.) In contrast, if there is significant rotation between absorption and emission, I∥ will be comparable to I195, and P will be close to zero. Polarization often is reported in milli-P units (1000×P), which for randomly oriented molecules will range between 0 and 500, because P will range between zero and one-half.
Polarization also may be described using other equivalent quantities, such as anisotropy. The relationship between anisotropy and intensity is expressed by the following equation:
Here, r is the anisotropy. Polarization and anisotropy include the same information, although anisotropy may be more simply expressed for systems containing more than one luminophore. In the description and claims that follow, these terms may be used interchangeably, and a generic reference to one should be understood to imply a generic reference to the other.
The relationship between polarization and rotation is expressed by the Perrin equation:
Here, P0 is the polarization in the absence of molecular motion (intrinsic polarization), τ is the luminescence lifetime (inverse decay rate) as described above, and τrot is the rotational correlation time (inverse rotational rate) as described above.
The Perrin equation shows that luminescence polarization assays are most sensitive when the luminescence lifetime and the rotational correlation time are similar. Rotational correlation time is proportional to molecular weight, increasing by about 1 nanosecond for each 2,400 dalton increase in molecular weight (for a spherical molecule). For shorter lifetime luminophores, such as fluorescein, which has a luminescence lifetime of roughly 4 nanoseconds, luminescence polarization assays are most sensitive for molecular weights less than about 40,000 daltons. For longer lifetime probes, such as Ru(bpy)2dcbpy (ruthenium 2,2′-dibipyridyl 4,4′-dicarboxyl-2,2′-bipyridine), which has a lifetime of roughly 400 nanoseconds, luminescence polarization assays are most sensitive for molecular weights between about 70,000 daltons and 4,000,000 daltons.
Luminescence polarization assays may be used in a variety of formats. In one format, the concentration of an analyte in solution can be measured by supplying a labeled tracer that competes with the analyte for a binding moiety, particularly a binding moiety larger than the labeled tracer. In this “competitive” format, the concentration of the analyte is inversely correlated with the enhancement of luminescence polarization in the light emitted by the tracer when it competitively binds the common moiety. In another format, the concentration of a target can be measured by supplying a labeled tracer that is capable of binding the target. In this case, the enhancement of polarization is a direct measure of the concentration of target. The target further may be, for example, an activated receptor, where activation can be indirectly measured by the directly measured concentration of a generated molecule or by its binding to labeled tracer per se.
C. Energy Transfer Assays
Energy transfer is the transfer of luminescence energy from a donor luminophore to an acceptor without emission by the donor. In energy transfer assays, a donor luminophore is excited from a ground state into an excited state by absorption of a photon. If the donor luminophore is sufficiently close to an acceptor, excited-state energy may be transferred from the donor to the acceptor, causing donor luminescence to decrease and acceptor luminescence to increase (if the acceptor is luminescent). The efficiency of this transfer is very sensitive to the separation R between donor and acceptor, decaying as 1/R−6. Energy transfer assays use energy transfer to monitor the proximity of donor and acceptor, which in turn may be used to monitor the presence or activity of an analyte, among others.
Energy transfer assays may focus on an increase in energy transfer as donor and acceptor are brought into proximity. These assays may be used to monitor binding, as between two molecules X and Y to form a complex X:Y. Here, colon (:) represents a noncovalent interaction. In these assays, one molecule is labeled with a donor D, and the other molecule is labeled with an acceptor A, such that the interaction between X and Y is not altered appreciably. Independently, D and A may be covalently attached to X and Y, or covalently attached to binding partners of X and Y.
Energy transfer assays also may focus on a decrease in energy transfer as donor and acceptor are separated. These assays may be used to monitor cleavage, as by hydrolytic digestion of doubly labeled substrates (peptides, nucleic acids). In one application, two portions of a polypeptide are labeled with D and A, so that cleavage of the polypeptide by a protease such as an endopeptidase will separate D and A and thereby reduce energy transfer. In another application, two portions of a nucleic acid are labeled with D and A, so that cleave by a nuclease such as a restriction enzyme will separate D and A and thereby reduce energy transfer.
Energy transfer between D and A may be monitored in various ways. For example, energy transfer may be monitored by observing an energy-transfer induced decrease in the emission intensity of D and increase in the emission intensity of A (if A is a luminophore). Energy transfer also may be monitored by observing an energy-transfer induced decrease in the lifetime of D and increase in the apparent lifetime of A.
In a preferred mode, a long-lifetime luminophore is used as a donor, and a short-lifetime luminophore is used as an acceptor. Suitable long-lifetime luminophores include metal-ligand complexes containing ruthenium, osmium, etc., and lanthanide chelates containing europium, terbium, etc. In time-gated assays, the donor is excited using a flash of light having a wavelength near the excitation maximum of D. Next, there is a brief wait, so that electronic transients and/or short-lifetime background luminescence can decay. Finally, donor and/or acceptor luminescence intensity is detected and integrated. In frequency-domain assays, the donor is excited using time-modulated light, and the phase and/or modulation of the donor and/or acceptor emission is monitored relative to the phase and/or modulation of the excitation light. In both assays, donor luminescence is reduced if there is energy transfer, and acceptor luminescence is observed only if there is energy transfer.
This example shows assays with improved signals, signal-to-noise ratios, and/or signal-to-background ratios.
Signal may be enhanced in several ways, including (1) using a high color temperature light source, such as a xenon arc lamp, in a continuous illumination mode, (2) using a dichroic or multi-dichroic beamsplitter, and/or (3) using a sample holder whose shape is “matched” to the shape of the optical beam of the instrument, especially if the sample holder is elevated to bring the sample closer to a detector. The high color temperature light source increases the number of usable photons, which is important because the lower limit of the signal-to-noise ratio is set by the square root of the total number of photons collected in the measurement. These enhancements are described in more detail in the following U.S. patent applications, which are incorporated herein by reference: Ser. Nos. 09/349,733, filed Jul. 8, 1999; 09/478,819, filed Jan. 5, 2000; and Ser. No. 09/494,407, filed Jan. 28, 2000.
Signal-to-noise ratios can be enhanced at least in part by increasing signals, for example, by using the techniques described in the previous paragraph.
Signal-to-background ratios can be enhanced in several ways, including (1) using confocal optical systems having a sensed volume to avoid luminescence from the microplate walls, (2) selecting a microplate or other substrate that increases the signal and reduces the luminescent background from materials in the microplate, (3) selecting the light sources, luminescence filters, optics, signal collection electronics, and mechanical system used in the luminescence detection optical system for maximum signal-to-background ratio, and (4) utilizing signal processing, background subtraction, and luminescence lifetime techniques, particularly FLAMe™ methodology for background reduction, as described below. These enhancements are described in more detail in the following U.S. patent and U.S. patent applications, which are incorporated herein by reference: U.S. Pat. No. 6,071,748, issued Apr. 17, 1998; Ser. Nos. 09/349,733, filed Jul. 8, 1999; Ser. No. 09/478,819, filed Jan. 5, 2000; and Ser. No. 09/494,407, filed Jan. 28, 2000.
This example shows mechanisms for increasing the change in polarization that accompanies a change in binding, so that the change in binding can be measured more easily. These mechanisms may be used in any of the assays described here involving luminescently labeled species, such as labeled cyclic nucleotides and labeled nonhydrolyzable GTP analogs, among others.
The change in polarization upon binding can be increased by making any linker between the luminophore and the labeled species (e.g., the cyclic nucleotide or GTP analog) as short and/or rigid as possible, while maintaining relevant substrate properties for the enzymes involved in the assay. Short and/or rigid linkers will restrict luminophore motion relative to the labeled species, reducing the “propeller effect” so that the luminophore more accurately reports the motion of both the free and bound labeled species. The rigidity of the linker may be increased by avoiding using hexanoic acid linkers, which typically are long and flexible, and by using cyclic linkers and amide groups in place of methylene groups, among other mechanisms.
The change in polarization upon binding also can be increased by including an appropriately positioned energy transfer acceptor on the binding partner, so that energy transfer will occur from the luminophore to the acceptor upon incorporation. Such energy transfer will shorten the lifetime of the luminophore, thereby increasing its polarization (because polarization varies inversely with lifetime, all else being equal).
The change in polarization upon binding also can be increased by decreasing the mobility of the binding partner for the labeled species. Mobility can be decreased by increasing the size of the binding partner, either directly or by forming a complex with a mass label. Suitable mass labels include other molecules and beads, among others. The use of mass labels is described in detail in U.S. patent application Ser. No. 09/768,742, filed Jan. 23, 2001, which is incorporated herein by reference. Mobility also can be decreased by attaching the binding partner to a surface, such as the surface of a sample holder. Attachment to other molecules, beads, and/or surfaces may be accomplished using any of a number of well-known reactive groups.
The assays provided by the invention may have advantages over prior assays for detecting molecular modifications. The existence and/or identity of these advantages will depend on such as (but not always requiring) one or more of the following. First, they may be used without radioactivity. Second, they may be homogenous, so that they do not require physical separation steps or wash steps. Third, they may have stable endpoints, so that results are relatively insensitive to the timing of any measurement or detection steps. Fourth, they may be sensitive, so that picomolar amounts of cyclic nucleotides may be detected. Fifth, they may be used with solution and cell-based samples.
The disclosure set forth above encompasses multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious and directed to one of the inventions. These claims may refer to “an” element or “a first” element or the equivalent thereof; such claims should be understood to include incorporation of one or more such elements, neither requiring nor excluding two or more such elements. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed through amendment of the present claims or through presentation of new claims in this or a related application. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.
Huang, Wei, Hoekstra, Merl F., Sportsman, J. Richard, Terpetschnig, Ewald A.
| Patent | Priority | Assignee | Title |
| 10324041, | Dec 21 2016 | ABBOTT JAPAN CO , LTD | Optical imaging system using lateral illumination for digital assays |
| 10335791, | Jan 18 2017 | THE KATHOLIEKE UNIVERSITEIT LEUVEN KU LEUVEN | Methods and devices for sample analysis |
| 11016053, | Oct 05 2016 | Abbott Laboratories | Devices and methods for sample analysis |
| 11022598, | Apr 03 2015 | Abbott Laboratories | Devices and methods for sample analysis |
| 11047854, | Feb 06 2017 | ABBOTT JAPAN CO LTD | Methods for reducing noise in signal-generating digital assays |
| 11073481, | Dec 21 2016 | Abbott Japan LLC | Optical imaging system using lateral illumination for digital assays |
| 11198129, | Oct 05 2016 | Abbott Laboratories | Devices and methods for sample analysis |
| 11369963, | Oct 05 2016 | Abbott Laboratories | Devices and methods for sample analysis |
| 11578358, | Sep 15 2016 | Abbott Laboratories | Devices and methods for sample analysis |
| 11633738, | Apr 03 2015 | Abbott Laboratories | Devices and methods for sample analysis |
| 11635387, | Dec 21 2016 | ABBOTT JAPAN CO., LTD | Optical imaging system using lateral illumination for digital assays |
| 11697117, | Jan 18 2017 | Abbott Laboratories; The Katholieke Universiteit Leuven | Methods and devices for sample analysis |
| 7445894, | May 03 2002 | Molecular Probes, Inc | Compositions and methods for detection and isolation of phosphorylated molecules |
| 7632651, | Sep 15 1997 | MOLECULAR DEVICES, INC | Molecular modification assays |
| 7674600, | Dec 15 2006 | Roche Palo Alto LLC | Assays for measuring phosphate modification enzyme activity |
| 7745142, | Sep 15 1997 | MOLECULAR DEVICES, INC | Molecular modification assays |
| 7776533, | May 03 2002 | Life Technologies Corporation | Compositions and methods for detection and isolation of phosphorylated molecules |
| 7901901, | Dec 15 2006 | Roche Palo Alto LLC | Assays for measuring phosphate modification enzyme activity |
| 9580745, | Nov 04 2005 | CLONDIAG GMBH | Method and device for the detection of molecular interactions |
| Patent | Priority | Assignee | Title |
| 3135793, | |||
| 3214377, | |||
| 3504052, | |||
| 3530049, | |||
| 3901654, | |||
| 3925162, | |||
| 3966556, | May 14 1971 | Syva Company | Compounds for enzyme amplification assay methadone analogs |
| 3996345, | Aug 12 1974 | Syva Company | Fluorescence quenching with immunological pairs in immunoassays |
| 4163780, | Mar 30 1977 | Kirin-Seagram Limited | KS-2-A |
| 4181654, | Jul 24 1978 | The United States of America as represented by the United States | 2,3-Dihydroxybenzoic acid amides of tetraazaalkanes and tetaaza cycloalkanes |
| 4199559, | Aug 12 1974 | Dade Behring Marburg GmbH | Fluorescence quenching with immunological pairs in immunoassays |
| 4205952, | May 31 1976 | Technion Research & Development Foundation Ltd. | Specific binding assay method and reagent means |
| 4220722, | Feb 10 1978 | Dade Behring Marburg GmbH | Method for conjugating to polyamino compounds employing haloacyl groups and compositions prepared thereby |
| 4238195, | Jan 18 1979 | Miles Laboratories, Inc. | Fluorescer-labeled specific binding assays |
| 4238395, | Jul 24 1978 | Miles Laboratories, Inc. | Dimethyl 7-[ω-N-(phthalimido)alkyl]aminonaphthalene-1,2-dicarboxylates |
| 4240751, | Nov 09 1978 | Akzona Incorporated | Method and apparatus for specific binding substances |
| 4257774, | Jul 16 1979 | BIOWHITTAKER, INC A CORP OF DELAWARE | Intercalation inhibition assay for compounds that interact with DNA or RNA |
| 4277437, | Apr 05 1978 | Dade Behring Marburg GmbH | Kit for carrying out chemically induced fluorescence immunoassay |
| 4280815, | Jun 18 1979 | Technicon Instruments Corporation | Electrochemiluminescent immunoassay and apparatus therefor |
| 4293310, | Mar 14 1980 | University of Pittsburgh | Photoelectrochemical immunoassay |
| 4318846, | Sep 07 1979 | Dade Behring Marburg GmbH | Novel ether substituted fluorescein polyamino acid compounds as fluorescers and quenchers |
| 4352395, | Nov 28 1980 | MARATHON OIL COMPANY, AN OH CORP | Process for selectively reducing the permeability of a subterranean formation |
| 4352751, | Sep 10 1979 | WIEDER, IRWIN | Species-linked diamine triacetic acids and their chelates |
| 4363759, | Apr 28 1975 | Miles Laboratories, Inc. | Chemiluminescent-labeled haptens and antigens |
| 4372745, | Dec 19 1979 | SCHIAPPARELLI BIOSYSTEMS, INC | Chemical luminescence amplification substrate system for immunochemistry involving microencapsulated fluorescer |
| 4374120, | Mar 08 1979 | Wallac Oy | Fluorescence spectroscopy assay means with fluorescent chelate of a lanthanide |
| 4378344, | Sep 28 1979 | Ventrex Laboratories, Inc. | Method and apparatus for performing multiple, simultaneous in vitro diagnostic tests using a solid phase system |
| 4412064, | Aug 09 1982 | Armco Inc. | Metal atom containing epoxy resins |
| 4419453, | Sep 28 1981 | The Dow Chemical Company | Immunological agglutination assays with dyed or colored latex and kits |
| 4425427, | Feb 16 1979 | VITAL SCIENTIFIC N V | Simultaneous, kinetic, spectrophotometric analysis of blood serum for multiple components |
| 4432907, | Sep 10 1979 | WIEDER, IRWIN | Diamine acid fluorescent chelates |
| 4459360, | Oct 05 1981 | HITACHI CHEMICAL DIAGNOSTICS, INC | Multiple-component binding assay system and method of making and using it |
| 4490216, | Feb 03 1983 | Molecular Devices Corporation | Lipid membrane electroanalytical elements and method of analysis therewith |
| 4492762, | Jul 30 1980 | Abbott Laboratories | Fluorescent polarization immunoassays |
| 4514508, | Jul 06 1982 | Biond Inc. | Assaying for a multiplicity of antigens or antibodies with a detection compound |
| 4542104, | Apr 06 1983 | The Board of Trustees of the Leland Stanford Jr. Univ. | Phycobiliprotein fluorescent conjugates |
| 4547527, | Dec 10 1984 | ARCO CHEMICAL TECHNOLOGY, L P A PARTNERSHIP OF DE | Impregnation suspension system for expandable styrene/maleic anhydride particles |
| 4565790, | Apr 30 1981 | Wallac Oy | Method for fluorescence spectroscopic determination of a biologically active substance |
| 4587223, | Sep 13 1982 | Wallac Oy | Method for quantitative determination of a biospecific affinity reaction |
| 4637988, | Jul 01 1981 | CLINICAL DIAGNOSTIC SYSTEMS INC | Fluorescent labels for immunoassay |
| 4652440, | May 03 1984 | Method of stably radiolabeling antibodies with technetium and rhenium | |
| 4656127, | Apr 22 1983 | AMERSHAM INTERNATIONAL PLC, WHITE LION ROAD, AMERSHAM, BUCKINGHAMSHIRE, HP7 9LL, ENGLAND | Method of detecting mutations in DNA and RNA |
| 4659839, | Oct 10 1984 | MALLINCKRODT MEDICAL, INC , A DE CORP | Coupling agents for radiolabeled antibody fragments |
| 4670572, | Jul 01 1981 | CLINICAL DIAGNOSTIC SYSTEMS INC | Phenolic fluorescent labels |
| 4687732, | Jun 10 1983 | YALE UNIVERSITY, A STATE OF CONNECTICUT | Visualization polymers and their application to diagnostic medicine |
| 4687747, | Jul 02 1984 | BYK-SANGTEC DIAGNOSTICA GMBH & CO KG | Phenanthridinium ester as a labelling compound in luminometric immunoassay |
| 4699978, | Jan 18 1985 | The Trustees of Columbia University in the City of New York | Site-specific chiral ruthenium (II) and cobalt (III) antitumor agents |
| 4704353, | Apr 27 1984 | MOLECULAR DEVICES CORPORATION, A CORP OF CA | Photoresponsive redox detection and discrimination |
| 4707440, | Jan 30 1984 | ENZO DIAGNOSTICS, INC , 40 OAK DRIVE, SYOSSET, NY 11791, A CORP OF NY | Nucleic acid hybridization assay and detectable molecules useful in such assay |
| 4711955, | Apr 17 1981 | Yale University | Modified nucleotides and methods of preparing and using same |
| 4721669, | Jan 18 1985 | The Trustees of Columbia University in the City of New York | Chemical probes for left-handed DNA and chiral metal complexes as Z-specific anti-tumor agents |
| 4741900, | Mar 09 1982 | CYTOGEN CORPORATION, A CORP OF DE | Antibody-metal ion complexes |
| 4745076, | Sep 17 1984 | Hoffmann-La Roche Inc. | Ruthenium complexes useful as carriers for immunologically active materials |
| 4751190, | Jul 22 1985 | ABBOTT LABORATORIES, A CORP OF IL | Fluorescence polarization immunoassay and reagents for use therein |
| 4761481, | Mar 18 1985 | DADE MICROSCAN INC | Substituted pyridine derivatives |
| 4772548, | Jan 30 1984 | ENZO DIAGNOSTICS, INC , 40 OAK DRIVE, SYOSSET, NY 11791, A CORP OF NY | Radioisotopicassay using isotope transfer to chelator-target recognition molecule conjugate |
| 4801804, | Sep 30 1986 | ZELTEX, INC | Method and apparatus for near infrared reflectance measurement of non-homogeneous materials |
| 4806488, | Sep 06 1985 | Dade Behring Marburg GmbH | Ligand-receptor assays employing squarate dye compositions |
| 4808541, | Mar 15 1983 | Wallac Oy | Determination method utilizing reagents covalently labelled with essentially non-fluorescent lanthanide chelates in combination with time-resolved fluorescence spectroscopy and the reagents to be used in the method |
| 4822733, | May 28 1985 | VYSIS, INC | Lifetime-resolved assay procedures |
| 4830786, | Feb 27 1986 | Dade Behring Marburg GmbH | Squaraine dyes |
| 4849330, | Apr 27 1984 | Molecular Devices Corporation | Photoresponsive redox detection and discrimination |
| 4851331, | May 16 1986 | Allied Corporation | Method and kit for polynucleotide assay including primer-dependant DNA polymerase |
| 4859609, | Apr 30 1986 | GENENTECH, INC , A CORP OF CA | Novel receptors for efficient determination of ligands and their antagonists or agonists |
| 4863849, | Jul 18 1985 | Biotage AB | Automatable process for sequencing nucleotide |
| 4863876, | Jan 15 1987 | Method of detecting and quantifying ligands in liquids via biotin-avidin-medicated fluorescence polarization | |
| 4876190, | Oct 21 1987 | Becton Dickinson & Company | Peridinin-chlorophyll complex as fluorescent label |
| 4876335, | Jun 30 1986 | Wakunaga Seiyaku Kabushiki Kaisha | Poly-labelled oligonucleotide derivative |
| 4883579, | Apr 27 1984 | Molecular Devices Corporation | Photoresponsive redox detection and discrimination |
| 4894347, | Sep 17 1987 | Agen Limited | Erythrocyte agglutination assay |
| 4915812, | Jun 20 1986 | Molecular Devices Corporation; MOLECULAR DEVICES CORPORATION, 3180 PORTER DRIVE, PALO ALTO, CA , 94303, A CORP OF CA | Zero volume cell |
| 4920195, | May 23 1985 | Fluorescent lanthanide chelates | |
| 4942127, | May 06 1988 | MOLECULAR DEVICES CORPORATION, A CORP OF CA | Polyredox couples in analyte determinations |
| 4943523, | Jan 30 1984 | ENZO DIAGNOSTICS, INC , 40 OAK DRIVE, SYOSSET, NY 11791, A CORP OF NY | Detectable molecules, method of preparation and use |
| 4946958, | Dec 11 1981 | University College Cardiff Consultants Limited | Chemiluminescent acridinium labelling compounds |
| 4956275, | Apr 14 1987 | Molecular Devices Corporation | Migratory detection immunoassay |
| 4962020, | Jul 12 1988 | President and Fellows of Harvard College | DNA sequencing |
| 4963658, | Sep 04 1987 | Molecular Devices Corporation | DNA detection method |
| 4966917, | Oct 25 1988 | Eastman Chemical Company | Methods for improving feed utilization and lactation in ruminant animals |
| 4978608, | Sep 04 1987 | Molecular Devices Corporation; MOLECULAR DEVICES CORPORATION, 3180 PORTER DRIVE, PALO ALTO, CA 94304 A CORP OF CA | DNA detection system |
| 5004806, | Oct 17 1988 | Molecular Devices Corporation | Nitrocellulose filtration to remove proteins from polynucleotides |
| 5011770, | Dec 15 1988 | MDS ANALYTICAL TECHNOLOGIES US INC | DNA detection method |
| 5030576, | Apr 30 1986 | Genentech, Inc. | Receptors for efficient determination of ligands and their antagonists or agonists |
| 5032677, | Nov 06 1987 | DADE MICROSCAN INC | Fluorescent poly(arylpyridine) rare earth chelates |
| 5055578, | May 30 1989 | DADE MICROSCAN INC | Fluorescent poly(arylpyridine) rare earth chelates |
| 5077037, | Aug 03 1990 | MALLINCKRODT MEDICAL, INC , A DE CORP | Novel compositions for magnetic resonance imaging |
| 5086002, | Sep 07 1987 | AGEN BIOMEDICAL, LTD | Erythrocyte agglutination assay |
| 5104804, | Jun 04 1990 | Molecular Devices Corporation | Cell assay device used in a microphysiometer |
| 5106957, | May 30 1989 | DADE MICROSCAN INC | Fluorescent poly(arylpyridine) rare earth chelates |
| 5112134, | Mar 01 1984 | MDS ANALYTICAL TECHNOLOGIES US INC | Single source multi-site photometric measurement system |
| 5116989, | Nov 06 1987 | DADE MICROSCAN INC | Fluorescent poly(arylpyridine) rare earth chelates |
| 5120644, | Apr 01 1986 | Wako Pure Chemical Industries, Ltd | Peptide derivatives and activity measuring method of physiologically active substances using the same as substrates |
| 5124457, | May 21 1986 | Abbott Laboratories | Phencyclidine and phencyclidine metabolites assay, tracers, immunogens and antibodies |
| 5141852, | Dec 12 1988 | MILES INC , ELKHART, IN A CORP OF IN | Assay of protein kinases with peptide substrates |
| 5155212, | May 21 1986 | ABBOTT LABORATORIES, A CORP OF ILLINOIS | Phencyclidine and phencyclidine metabolites assay, tracers, immunogens, antibodies and reagent kit |
| 5180828, | Feb 09 1990 | MOLECULAR DEVICES, INC | Chromophoric reagents for incorporation of biotin or other haptens into macromolecules |
| 5202423, | Jul 08 1988 | Wallac Oy | Terpyridine derivatives |
| 5216134, | Oct 23 1989 | Wallac Oy | Spectrofluorometric method and compounds that are of value for the method |
| 5221605, | Oct 31 1984 | Bioveris Corporation | Luminescent metal chelate labels and means for detection |
| 5225543, | Mar 28 1991 | American Cyanamid Company | Receptors method for purification of G protein-linked |
| 5232858, | Apr 25 1989 | AVL Medical Instruments AG | Method for quantitative determination of at least one chemical parameter of a sample medium |
| 5235039, | Jun 10 1991 | Eli Lilly and Company | Substrates for HIV protease |
| 5238808, | Oct 31 1984 | Bioveris Corporation | Luminescent metal chelate labels and means for detection |
| 5245038, | Nov 06 1987 | DADE MICROSCAN INC | Fluorescent poly(arylpyridine) rare earth chelates |
| 5246867, | Jan 17 1992 | LAKOWICZ, JOSEPH R , PH D | Determination and quantification of saccharides by luminescence lifetimes and energy transfer |
| 5252293, | Jan 17 1989 | MOLECULAR DEVICES, INC | Analytical slide with porous filter membrane |
| 5252462, | May 05 1988 | Pharmacia AB; Wallac Oy | Enzyme activity determinations method characterized by the using of substrates whose fluorescent properties differs those of the converted products |
| 5252740, | Nov 06 1987 | DADE MICROSCAN INC | Fluorescent poly(arylpyridine) rare earth chelates |
| 5256535, | Oct 17 1986 | Wallac Oy | Hybridization assay and means to be used in the assay |
| 5258512, | Feb 28 1989 | Abbott Laboratories | Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies |
| 5260200, | Jan 18 1991 | Joslin Diabetes Center, Inc. | Isolated DNA encoding an insulin receptor substrate |
| 5260441, | Jul 14 1986 | Abbott Laboratories | Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies |
| 5270171, | Mar 06 1987 | Cancer-associated SCM-recognition factor, preparation and method of use | |
| 5274113, | Nov 01 1991 | Molecular Probes, Inc.; MOLECULAR PROBES, INC A CORP OF OREGON | Long wavelength chemically reactive dipyrrometheneboron difluoride dyes and conjugates |
| 5278048, | Oct 21 1988 | Molecular Devices Corporation | Methods for detecting the effect of cell affecting agents on living cells |
| 5279943, | Aug 02 1985 | Compagnie Oris Industrie | Homogeneous process for the detection and/or determination by luminescence of an analyte in a medium in which it may be present |
| 5283173, | Jan 24 1990 | The Research Foundation of State University of New York | System to detect protein-protein interactions |
| 5302509, | Aug 14 1989 | BECKMAN INSTRUMENTS, INC , A CORP OF DELAWARE | Method for sequencing polynucleotides |
| 5310687, | Oct 31 1984 | Bioveris Corporation | Luminescent metal chelate labels and means for detection |
| 5312986, | Mar 03 1989 | Willi Moller AG | Hexacarboxylic acid hexa-amides which form liphophilic complexes with magnesium ions, corresponding magnesium complexes, and test devices and ion selective parts containing such hexacarboxylic acid hexa-amides |
| 5315015, | Nov 10 1992 | Roche Diagnostics Corporation | Compounds having improved fluorescence in fluorescence polarization immunoassays and immunoassays utilizing same |
| 5316909, | Mar 15 1991 | Wallac Oy | Method for increasing fluorescence |
| 5324825, | Jul 08 1988 | Wallac Oy | Terpyridine derivatives |
| 5326692, | May 13 1992 | BIOSITE, INC | Fluorescent microparticles with controllable enhanced stokes shift |
| 5340714, | May 08 1992 | MONITOR DIAGNOSTICS, INC A CORP OF UTAH | Use of nonmetallic tetrapyrrole molecules and novel signal solutions in chemiluminescent reactions and assays |
| 5340716, | Jun 20 1991 | Siemens Healthcare Diagnostics Products GmbH | Assay method utilizing photoactivated chemiluminescent label |
| 5341215, | Jun 08 1991 | Koninklijke Philips Electronics N V | Method and apparatus for detecting the presence and/or concentration of biomolecules |
| 5369108, | Oct 04 1991 | Sloan-Kettering Institute for Cancer Research; SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH A CORPORATION OF NY; TRUSTEES OF COLUMBIA UNIVERSITY, THE A CORPORATION OF NY | Potent inducers of terminal differentiation and methods of use thereof |
| 5374531, | Mar 22 1993 | CREDITANSTALT BANKVEREIN | Immunoassay for determination of cells |
| 5384108, | Apr 24 1989 | MALLINCKRODT MEDICAL, INC , A DE CORP | Magnetic resonance imaging agents |
| 5393659, | Dec 02 1988 | Dade Behring Marburg GmbH | Agent for immunochemical tests which contains polymers containing carboxyl groups |
| 5403928, | May 15 1990 | Diatron Corporation; DIATRON CORPORATION A CORPORATION OF CA | Fluorescent marker components and fluorescent probes |
| 5409666, | Aug 08 1991 | Terumo Cardiovascular Systems Corporation | Sensors and methods for sensing |
| 5409835, | Dec 30 1992 | LAKOWICZ, JOSEPH R | Long-wavelength fluorescent probe compounds for calcium ions and their use in ratiometrically measuring calcium ion concentrations |
| 5433896, | May 20 1994 | Molecular Probes, Inc.; Molecular Probes, Inc | Dibenzopyrrometheneboron difluoride dyes |
| 5434088, | Sep 14 1990 | Tosoh Corporation | Method of and kit for energy transfer immunoassay with colloidal particles |
| 5443791, | Apr 06 1990 | Applied Biosystems, LLC | Automated molecular biology laboratory |
| 5445935, | Nov 23 1992 | R-P TECHNOLOGIES | Quantitative detection of macromolecules with fluorescent oligonucleotides |
| 5453356, | Oct 31 1984 | Bioveris Corporation | Luminescent metal chelate labels and means for detection |
| 5457186, | Oct 13 1993 | Wallac Oy | Luminescent lanthanide chelates with decreased non-radiative energy loss |
| 5464607, | Dec 10 1991 | Dibra S.p.A. | 5,5'-/(1,3-propanediyl) bis-/imino(2-oxo-2,1-ethanediyl)acetylimino/bis(2,4,6-triiodo-1,3-benzen edicarboxylamides), and contrast media containing them |
| 5466578, | Apr 09 1992 | The United States of America as represented by the Secretary of the Navy; NAVY, THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE | Surfactant-enhanced light emission- or absorbance-based binding assays for polynucleic acids |
| 5478754, | Mar 04 1992 | ABBOTT LABOROTORIES | Determination of glycated hemoglobin by fluorescence quenching |
| 5482699, | Mar 27 1991 | AMERSHAM HEALTH SALUTAR INC | Multinuclear complexes for x-ray imaging |
| 5494793, | Dec 15 1986 | S R ONE, LIMITED | Monomeric phthalocyanine reagents |
| 5501956, | Mar 23 1990 | Molecular Devices Corporation | Polyredox couples in analyte determinations |
| 5512493, | Jul 13 1990 | CIS Bio International | Method of amplifying the emission signal of a luminescent compound |
| 5518900, | Jan 15 1993 | Beckman Coulter, Inc | Method for generating single-stranded DNA molecules |
| 5525479, | Jun 17 1992 | MERCK & CO , INC | Fluorescence assay of Ras farnesyl protein transferase |
| 5527684, | Jan 28 1991 | CIS Bio International | Method of measuring the luminescence emitted in a luminescent assay |
| 5527688, | Apr 08 1994 | Pierce Biotechnology, Inc | Rapid assays for protein kinase activity |
| 5531978, | Jul 16 1987 | GE HEALTHCARE AS | Aminopolycarboxylic acids and derivatives thereof |
| 5538858, | Apr 08 1994 | Pierce Biotechnology, Inc | Rapid assay for radioactive determination of protein kinase activity |
| 5541113, | Sep 22 1993 | Beckman Instruments, Inc. | Method for detecting an analyte using an electrochemical luminescent transition metal label |
| 5554749, | Jan 14 1994 | Mallinckrodt Inc | Functionalized macrocyclic ligands for imaging applications |
| 5561051, | Jun 14 1994 | BASF Aktiengesellschaft | Screen for inhibitors of chitinase |
| 5561052, | Jun 18 1992 | Process for detecting oxidized lipids and process for forming oxidized lipids | |
| 5567302, | Jun 07 1995 | Molecular Devices Corporation | Electrochemical system for rapid detection of biochemical agents that catalyze a redox potential change |
| 5571684, | Nov 07 1994 | LITMUS CONCEPTS, INC | Assay for proline iminopeptidase and other hydrolytic activities |
| 5571897, | Dec 05 1991 | Wallac Oy | Luminescent lanthanide chelates |
| 5573752, | Apr 08 1994 | Bracco International B.V. | Aromatic amide compounds and metal chelates thereof |
| 5591581, | Apr 30 1986 | Bioveris Corporation | Electrochemiluminescent rhenium moieties and methods for their use |
| 5593867, | Apr 18 1994 | Becton, Dickinson and Company | Fluorerscence polarization detection of nucleic acid amplication |
| 5599681, | Apr 10 1992 | DANA-FARBER CANCER INSTITUTE, INC | Activation-state-specific phosphoprotein immunodetection |
| 5610075, | Jan 17 1995 | Bioveris Corporation | Competitive electrochemiluminescence assays for endotoxins using a ruthenium label |
| 5610287, | Dec 06 1993 | Beckman Coulter, Inc | Method for immobilizing nucleic acid molecules |
| 5614368, | Feb 09 1990 | MOLECULAR DEVICES, INC | Chromophoric reagents for incorporation of biotin or other haptens into macromolecules |
| 5616312, | Mar 10 1995 | Mallinckrodt Medical, Inc. | Thiol ligands and complexes for X-ray imaging |
| 5621075, | Jan 18 1991 | Joslin Diabetes Center, Inc. | Insulin receptor substrate |
| 5622821, | Jun 29 1994 | Regents of the University of California, The | Luminescent lanthanide chelates and methods of use |
| 5624847, | May 03 1991 | LAKOWICZ, JOSEPH R | Method for optically measuring chemical analytes |
| 5629157, | Apr 22 1993 | Dade Behring Marburg GmbH | Double receptor polynucleotide assay method |
| 5631127, | Jan 22 1992 | AXIS-SHIELD ASA | Enzymatic assay for homocysteine and a kit therefor |
| 5631169, | Jan 27 1992 | LAKOWICZ, JOSEPH R | Fluorescent energy transfer immunoassay |
| 5632982, | Jun 07 1994 | BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY, THE | Cytotoxic enhancement of TNF with copper |
| 5637463, | May 04 1995 | Hoffmann-La Roche Inc.; Hoffmann-La Roche Inc | Method to detect protein-protein interactions |
| 5637509, | Sep 24 1987 | Wallac Oy | Modulated homogeneous fluorescence biospecific affinity assay employing fluorescing lanthanide chelates as covalent labels |
| 5639599, | Jun 08 1992 | Hologic, Inc; Biolucent, LLC; Cytyc Corporation; CYTYC SURGICAL PRODUCTS, LIMITED PARTNERSHIP; SUROS SURGICAL SYSTEMS, INC ; Third Wave Technologies, INC; Gen-Probe Incorporated | Amplification of nucleic acids from mononuclear cells using iron complexing and other agents |
| 5639615, | Jun 29 1994 | The Regents of the University of California | Luminescent lanthanide chelates and methods of use |
| 5641633, | Nov 15 1995 | Becton, Dickinson and Company | Fluorescence polarization detection of nucleic acids |
| 5641878, | May 15 1991 | Diatron Corporation | Porphyrin, azaporphyrin, and related fluorescent dyes free of aggregation and serum binding |
| 5645800, | Oct 31 1991 | Beckman Coulter, Inc | Specimen processing and analyzing systems with associated fluid dispensing apparatus |
| 5648269, | May 03 1991 | Joseph R., Lakowicz | pH and pCO2 sensing by luminescent lifetimes and energy transfer |
| 5656254, | May 18 1994 | Bracco Suisse SA | Polyaza heteroatom-bearing ligands and metal complexes thereof for imaging or radiotherapy |
| 5656433, | Jun 29 1994 | The Regents of the University of California | Luminescent lanthanide chelates and methods of use |
| 5660991, | Oct 28 1994 | LAKOWICZ, JOSEPH R | Long lifetime anisotropy (polarization) probes for clinical chemistry, immunoassays, affinity assays and biomedical research |
| 5668110, | Jun 07 1995 | AFFYMAX, INC | Peptides and compounds that bind to the IL-5 receptor |
| 5670113, | Dec 20 1991 | Merck Sharp & Dohme Corp | Automated analysis equipment and assay method for detecting cell surface protein and/or cytoplasmic receptor function using same |
| 5676943, | Aug 12 1993 | NEUROTECH USA, INC | Compositions and methods for the delivery of biologically active molecules using genetically altered cells contained in biocompatible immunoisolatory capsules |
| 5677196, | May 18 1993 | University of Utah Research Foundation | Apparatus and methods for multi-analyte homogeneous fluoro-immunoassays |
| 5677199, | May 15 1990 | Diatron Corporation | Fluorescent marker components and fluorescent probes |
| 5677280, | Jun 07 1995 | AFFYMAX, INC | Peptides and compounds that bind to the IL-5 receptor |
| 5683983, | Jun 07 1995 | AFFYMAX, INC | Peptides and compounds that bind to the IL-5 receptor |
| 5705045, | Aug 29 1995 | LG Electronics Inc | Multi-biosensor for GPT and got activity |
| 5707813, | May 15 1990 | Diatron Corporation | Nucleic acid probes and methods |
| 5711915, | Mar 18 1992 | Siemens Healthcare Diagnostics GmbH | Optical solid-phase biosensor based on polyionic layers labelled with fluorescent dyes |
| 5723304, | Aug 03 1992 | Marconi Caswell Limited | Immunological detection using two detectable labels |
| 5731147, | Oct 31 1984 | Bioveris Corporation | Luminescent metal chelate labels and means for detection |
| 5738825, | Jul 20 1993 | Unaxis Balzers Aktiengesellschaft | Optical biosensor matrix |
| 5739001, | Oct 29 1996 | NEN LIFE SCIENCE PRODUCTS, INC | Solid phase cell-based assay |
| 5741714, | Jul 16 1996 | Janssen Diagnostics, LLC | Detection of bound analyte by magnetic partitioning and masking |
| 5741715, | May 30 1995 | Roche Diagnostics Corporation | Quinidine immunoassay and reagents |
| 5744320, | Jun 07 1995 | Promega Corporation | Quenching reagents and assays for enzyme-mediated luminescence |
| 5750410, | Jan 26 1996 | Kyoto Dai-Ichi Kagaku Co., Ltd. | Method of and apparatus for immune analysis |
| 5756292, | Nov 23 1992 | R-P Technologies, Inc. | Quantitative detection of macromolecules with fluorescent oligonucleotides |
| 5756304, | Jul 14 1995 | Molecular Solutions | Screening of microorganisms for bioremediation |
| 5760188, | Dec 31 1992 | Martin R&F Inc. | Marker for neurotensin receptor |
| 5762910, | May 23 1991 | IMARX THERAPEUTICS, INC | Liposoluble compounds useful as magnetic resonance imaging agents |
| 5763158, | Feb 06 1997 | The United States of America as represented by the Secretary of the Army | Detection of multiple antigens or antibodies |
| 5770455, | Jul 19 1993 | NEXUS BIOSYSTEMS, INC | Methods and apparatus for synthesizing labeled combinatorial chemistrylibraries |
| 5773257, | Oct 24 1989 | Agilent Technologies, Inc | Method for producing primed nucleic acid templates |
| 5783397, | Jun 12 1996 | Northeastern University | Screening natural samples for new therapeutic compounds using capillary electrophoresis |
| 5783687, | Mar 14 1990 | Regents of the University of California, The | Dyes designed for high sensitivity detection of double-stranded DNA |
| 5786139, | Dec 09 1994 | Life Technologies Corporation | Method and kit for detecting nucleic acid cleavage utilizing a covalently attached fluorescent tag |
| 5798085, | Feb 21 1996 | Biomerieux, Inc | Optical reader and sample card transport stations for biological sample testing machine |
| 5800778, | May 31 1995 | Biomerieux, Inc | Sealant for sample holder |
| 5800989, | Nov 15 1995 | Becton, Dickinson and Company | Method for detection of nucleic acid targets by amplification and fluorescence polarization |
| 5800996, | May 03 1996 | Applied Biosystems, LLC | Energy transfer dyes with enchanced fluorescence |
| 5801149, | Jun 19 1991 | JOSLIN DIABETES CENTER, INC | Inhibition of signal transduction molecules |
| 5804395, | Dec 01 1995 | The United States of America as represented by the Secretary of the Navy | Fluorescence polarization assays of enzymes and substrates therefore |
| 5807522, | Jun 17 1994 | LELAND STANFORD, JUNIOR UNIVERSITY OF, BOARD OF TRUSTEES, THE | Methods for fabricating microarrays of biological samples |
| 5811256, | Oct 10 1995 | Monell Chemical Senses Center | Method for measuring sensory irritation in vitro |
| 5820849, | Jul 04 1995 | Bayer Schering Pharma Aktiengesellschaft | Cascade polymer complexes, process for their production and pharamceutical agents containing said complexes |
| 5824517, | Jul 24 1995 | Bio Merieux | Method for amplifying nucleic acid sequences by strand displacement using DNA/RNA chimeric primers |
| 5824557, | Apr 02 1996 | Life Technologies Corporation | Method for detecting and quantitating nucleic acid impurities in biochemical preparations |
| 5824772, | Apr 04 1995 | Advanced Bioconcept Company | Fluorescent somatostatin |
| 5827653, | Sep 23 1993 | AstraZeneca UK Limited | Nucleic acid detection with energy transfer |
| 5830769, | Mar 18 1985 | NEN LIFE SCIENCE PRODUCTS, INC ; PERKINELMER LIFE SCIENCES, INC | Homogeneous fluorassay methods employing fluorescent background rejection and water-soluble rare earth metal chelates |
| 5846710, | Nov 02 1990 | ORCHID CELLMARK INC | Method for the detection of genetic diseases and gene sequence variations by single nucleotide primer extension |
| 5846722, | Oct 16 1996 | TELIK, INC | System to detect small molecule/peptide interaction |
| 5849547, | Jul 26 1993 | Bio Merieux | Method for nucleic acid amplification by transcription using displacement, and reagents and kit therefor |
| 5849794, | Jan 21 1994 | Ferring BV | Guanylhydrazones and their use to treat inflammatory conditions |
| 5852191, | Jun 07 1995 | Carnegie Mellon University | Rigidized monomethine cyanines |
| 5853999, | Mar 20 1987 | The Regents of the University of California | Methods of screening for agents that inhibit interaction between MHC class I antigens and a cell surface receptor |
| 5854008, | Apr 06 1992 | 5131 INVESTMENTS LTD | Europium and terbium chelators for the time-resolved fluorometric assays |
| 5858671, | Nov 01 1996 | The University of Iowa Research Foundation | Iterative and regenerative DNA sequencing method |
| 5858676, | Apr 18 1995 | Bioveris Corporation | Electrochemiluminescence of rare earth metal chelates |
| 5858805, | Jan 13 1997 | Roche Diagnostics Corporation | Assay for aminoglycoside antibiotics |
| 5859215, | Oct 25 1995 | Wallac Oy | Biospecific binding reactants labelled with luminescent lanthanide chelates and their use |
| 5861239, | Jun 30 1995 | Millennium Pharmaceuticals, Inc | Methods for identifying compounds that modulate mammalian tub protein activity |
| 5861262, | Sep 03 1993 | Oxis Isle of Man, Limited | Method of the specific immunoassay of human plasma glutathione peroxidase, kit for its implementation, oligopeptides and antibodies specific for the method |
| 5866335, | Jun 24 1994 | George W., Katsilometes | Preparation of derivatized 10,10'-substituted-9,9'-biacridine luminescent molecules and signal solutions |
| 5871713, | Dec 04 1991 | Guerbet S.A. | Macromolecular polyamine iodine-containing compound, process for its preparation and its use as a contrast agent |
| 5874214, | Apr 25 1995 | NEXUS BIOSYSTEMS, INC | Remotely programmable matrices with memories |
| 5880096, | Feb 02 1994 | AFFYMAX TECHNOLOGIES N V | Peptides and compounds that bind to the IL-1 receptor |
| 5880296, | May 10 1995 | Pierre Fabre Medicament | Method for preparing an optically pure benzofuran carboxylic acid and use thereof for preparing efaroxan |
| 5885779, | Sep 09 1997 | INTEROMEX BIOPHARMACEUTICALS, INC | Repressed trans-activator system for characterization of protein-protein interactions |
| 5888728, | Oct 17 1988 | MOLECULAR DEVICES, INC | Hapten derivatized capture membrane and diagnostic assays using such membrane |
| 5888819, | Mar 05 1991 | ORCHID CELLMARK INC | Method for determining nucleotide identity through primer extension |
| 5891621, | Sep 24 1997 | Merck & Co., Inc. | Metabolic pathway assay |
| 5891674, | Jun 19 1997 | Dupont Pharmaceuticals Company | Insulin receptor tyrosine kinase substrate |
| 5891696, | Aug 01 1997 | Life Technologies Corporation | Compositions for cytochrome P450 biotransformation reactions |
| 5897674, | Dec 29 1995 | POHANG IRON & STEEL CO , LTD ; Research Institute of Industrial Science & Technology | Method for manufacturing coal agglomerates for use in direct iron smelting reducing furnace |
| 5910574, | Aug 05 1994 | Genentech, Inc. | Human trk receptors and neurotrophic factor inhibitors |
| 5912137, | Jul 16 1996 | Regents of the University of California, The | Assays for protein kinases using fluorescent |
| 5914230, | Dec 22 1995 | Siemens Healthcare Diagnostics Products GmbH | Homogeneous amplification and detection of nucleic acids |
| 5945283, | Dec 17 1996 | Washington University | Methods and kits for nucleic acid analysis using fluorescence resonance energy transfer |
| 5948620, | Aug 05 1996 | Amersham International plc | Reverse two-hybrid system employing post-translation signal modulation |
| 5958694, | Oct 16 1997 | Caliper Technologies Corp. | Apparatus and methods for sequencing nucleic acids in microfluidic systems |
| 5962243, | Apr 27 1995 | Board of Regents, The University of Texas System | Methods for the identification of farnesyltransferase inhibitors |
| 5981180, | Oct 11 1995 | LUMINEX CORPORATION | Multiplexed analysis of clinical specimens apparatus and methods |
| 5981185, | May 05 1994 | Affymetrix, Inc | Oligonucleotide repeat arrays |
| 5985550, | Apr 22 1993 | Dade Behring Marburg GmbH | Double receptor polynucleotide assay method |
| 5989835, | Feb 27 1997 | CELLOMICS, INC | System for cell-based screening |
| 6004744, | Mar 05 1991 | ORCHID CELLMARK INC | Method for determining nucleotide identity through extension of immobilized primer |
| 6005113, | May 15 1996 | Molecular Probes, Inc. | Long wavelength dyes for infrared tracing |
| 6007984, | Nov 01 1989 | AstraZeneca UK Limited | Detection of DNA/RNA by fluorescence polarization |
| 6013431, | Feb 16 1990 | ORCHID CELLMARK INC | Method for determining specific nucleotide variations by primer extension in the presence of mixture of labeled nucleotides and terminators |
| 6013457, | Jun 24 1994 | Siemens Healthcare Diagnostics Products GmbH | Method for carrying out an immunoassay in a multiphase system |
| 6022708, | Feb 26 1998 | Genentech, Inc | Fused |
| 6025129, | Jun 07 1995 | IRORI TECHNOLOGIES, INC | Remotely programmable matrices with memories and uses thereof |
| 6037136, | Oct 24 1994 | Cold Spring Harbor Laboratory | Interactions between RaF proto-oncogenes and CDC25 phosphatases, and uses related thereto |
| 6045755, | Mar 10 1997 | Lion Bioscience AG | Apparatus and method for combinatorial chemistry synthesis |
| 6045996, | Oct 26 1993 | Affymetrix, Inc | Hybridization assays on oligonucleotide arrays |
| 6054557, | Apr 04 1995 | Advanced Bioconcept Company | Fluorescent peptides |
| 6071748, | Sep 20 1997 | MOLECULAR DEVICES, INC | Light detection device |
| 6137584, | Nov 27 1996 | Max-Planck-Gesellschaft Zur; Evotec BioSystems Aktiengesellschaft | Method and device for determining predetermined properties of target particles of a sample medium |
| 6451871, | Nov 25 1998 | Novartis AG | Methods of modifying surface characteristics |
| 6472141, | May 21 1999 | CALIPER TECHNOLOGIES CORP | Kinase assays using polycations |
| 6703498, | Jun 21 2001 | TAKARA BIO USA, INC | Water-soluble polymeric metal ion affinity compositions and methods for using the same |
| CA2099542, | |||
| EP137457, | |||
| EP178450, | |||
| EP204109, | |||
| EP242847, | |||
| EP259386, | |||
| EP278149, | |||
| EP312897, | |||
| EP317074, | |||
| EP382433, | |||
| EP457213, | |||
| EP578067, | |||
| EP639647, | |||
| EP648280, | |||
| EP650521, | |||
| EP678581, | |||
| EP774515, | |||
| EP774516, | |||
| GB2223096, | |||
| JP1143874, | |||
| JP1231898, | |||
| JP2295496, | |||
| JP5123196, | |||
| JP643159, | |||
| WO819, | |||
| WO6989, | |||
| WO6990, | |||
| WO6991, | |||
| WO11220, | |||
| WO14515, | |||
| WO23785, | |||
| WO42209, | |||
| WO47693, | |||
| WO48990, | |||
| WO48991, | |||
| WO55372, | |||
| WO66269, | |||
| WO75167, | |||
| WO75662, | |||
| WO75664, | |||
| WO8100261, | |||
| WO8101883, | |||
| WO8707385, | |||
| WO8806633, | |||
| WO9005301, | |||
| WO9113075, | |||
| WO9211039, | |||
| WO9215712, | |||
| WO9310461, | |||
| WO9319206, | |||
| WO9325672, | |||
| WO9512607, | |||
| WO9521271, | |||
| WO9603410, | |||
| WO9722719, | |||
| WO9735033, | |||
| WO9740104, | |||
| WO9745539, | |||
| WO9745739, | |||
| WO9801472, | |||
| WO9805962, | |||
| WO9812156, | |||
| WO9818956, | |||
| WO9823942, | |||
| WO9845481, | |||
| WO9859066, | |||
| WO9911774, | |||
| WO9923466, | |||
| WO9929894, | |||
| WO9931431, | |||
| WO9936779, | |||
| WO9960383, | |||
| WO9960385, |
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